DNA encoding mammalian neuropeptide FF (NPFF) receptors and uses thereof

ABSTRACT

This invention provides isolated nucleic acids encoding mammalian NPFF receptors, purified mammalian NPFF receptors, vectors comprising nucleic acid encoding mammalian NPFF receptors, cells comprising such vectors, antibodies directed to mammalian NPFF receptors, nucleic acid probes useful for detecting nucleic acid encoding mammalian NPFF receptors, antisense oligonucleotides complementary to unique sequences of nucleic acid encoding mammalian NPFF receptors, transgenic, nonhuman animals which express DNA encoding normal or mutant mammalian NPFF receptors, methods of isolating mammalian NPFF receptors, methods of treating an abnormality that is linked to the activity of the mammalian NPFF receptors, as well as methods of determining binding of compounds to mammalian NPFF receptors, methods of identifying agonists and antagonists of NPFF receptors, and agonists and antagonists so identified.

BACKGROUND OF THE INVENTION

[0001] This application is a continuation-in-part of U.S. Ser. No.09/161,113, filed Sep. 25, 1998 the contents of which are herebyincorporated by reference into the subject application.

[0002] Throughout this application, various publications are referencedin parentheses by author and year. Full citations for these referencesmay be found at the end of the specification immediately preceding thesequence listings and the claims. The disclosure of these publicationsin their entireties are hereby incorporated by reference into thisapplication to describe more fully the art to which this inventionpertains.

[0003] Neuroregulators comprise a diverse group of natural products thatsubserve or modulate communication in the nervous system. They include,but are not limited to, neuropeptides, amino acids, biogenic amines,lipids and lipid metabolites, and other metabolic byproducts. Many ofthese neuroregulator substances interact with specific cell surfacereceptors which transduce signals from the outside to the inside of thecell. G-protein coupled receptors (GPCRs) represent a major class ofcell surface receptors with which many neurotransmitters interact tomediate their effects. GPCRs are predicted to have sevenmembrane-spanning domains and are coupled to their effectors viaG-proteins linking receptor activation with intracellular biochemicalsequelae such as stimulation of adenylyl cyclase.

[0004] Neuropeptide FF (NPFF) is an octapeptide isolated from bovinebrain in 1985 by Yang and coworkers (1) using antibodies to themolluscan neuropeptide FMRFamide (FMRFa). FMRFamide-likeimmunoreactivity was observed in rat brain, spinal cord, and pituitary,suggesting the existence of mammalian homologs of the FMRFa family ofinvertebrate peptides. The isolation of NPFF, named for its N- andC-terminal phenylalanines (also called F8Famide) and a second mammalianpeptide, NPAF (also called A18Famide), confirmed the existence ofmammalian family of peptides sharing C-terminal sequence homology withFMRFa (1). Molecular cloning has revealed that NPFF and NPAF are encodedby the same gene and cleaved from a common precursor protein (2).Studies of the localization, radioligand binding, and function ofNPFF-like peptides (see below) indicate they are neuromodulatorypeptides whose effects are likely to be mediated by G protein-coupledreceptors (for review, see 3).

[0005] NPFF, also called “morphine modulating peptide”, is an endogenousmodulator of opioid systems with effects on morphine analgesia,tolerance, and withdrawal (for review see 3, 4). NPFF appears torepresent an endogenous “anti-opioid” system in the CNS acting atspecific, high-affinity receptors distinct from opiate receptors (5, 6).Endogenous NPFF has been suggested to play a role in morphine tolerance:agonists of NPFF precipitate “morphine abstinence syndrome” (i.e.symptoms of morphine withdrawal) in morphine-dependent animals (7, 8),while antagonists and anti-NPFF IgG restore morphine sensitivity andameliorate symptoms of withdrawal (9-12). NPFF antagonists potentiallycould be useful as therapeutic agents to prevent the development ofmorphine tolerance, and to treat opiate addiction. NPFF has also beensuggested to participate in the regulation of pain threshold, showingboth “anti-opiate” effects and analgesic effects depending on testsystem and route of administration (for review, see 4). As ananti-opiate, NPFF has been shown to inhibit morphine- and stress-inducedanalgesia (1, 13, 14, 15), whereas anti-NPFF IgG (which blocks thebiological activity of NPFF) potentiates these two phenomena (16, 17).An NPFF antagonist may be clinically useful in potentiating theanalgesic effects of morphine, allowing use of lower doses without thedevelopment of tolerance. NPFF agonists may also exhibit analgesicactivity in some model systems (14, 18, 19). The analgesia elicited byNPFF is typically sensitive to naloxone, indicating that it is mediatedby release of endogenous opioid peptides (19, 20). The interaction ofNPFF and opioid systems in regulating pain pathways is thus complex andmay involve multiple mechanisms and sites of action. NPFF has additionalbiological activities in accord with its pattern of expression in thenervous system.

[0006] NPFF peptide localization in rat CNS was examined using specificantibodies ((21-23); see also (3)). The highest levels of NPFF are foundin spinal cord and posterior pituitary; pituitary NPFF is believed tooriginate in the hypothalamus. In the brain, immunoreactive cell bodiesare found in two major cell groups: medial hypothalamus (betweendorsomedial and ventromedial) and nucleus of the solitary tract.Immunoreactive fibers are observed in lateral septal nucleus, amygdala,hypothalamus, nucleus of solitary tract, ventral medulla, trigeminalcomplex, and dorsal horn of spinal cord. This localization pattern isconsistent with a role for NPFF in sensory processing and modulation ofopioid systems. In addition, its presence in the hypothalamus and otherlimbic structures could subserve roles in the regulation of appetitiveand affective states. In the periphery, NPFF-like immunoreactivity (aswell as NPFF binding) has been observed in the heart (24). In addition,injection of NPFF raises blood pressure in rats (24, 25). Theseobservations, combined with the colocalization of NPFF withcatecholaminergic neurons in the nucleus of the solitary tract (26),suggest that NPFF is involved in central and peripheral cardiovascularregulation.

[0007] The ability of NPFF peptides to modulate the opioid system raisedthe possibility that NPFF interacts directly with opiate receptors.However, radioligand binding assays using a tyrosine-substituted NPFFanalog [¹²⁵I]Y8Fa demonstrate that NPFF acts through specific highaffinity binding sites distinct from opiate receptors (27-30) that aresensitive to inhibition by guanine nucleotides (31). The latterobservation indicates that NPFF receptors are likely to belong to thesuperfamily of G protein-coupled receptors which share common structuralmotifs. However, no reports of cloning NPFF receptors have appeared asyet.

[0008] To address the issue of potential degradation of the peptideradioligand, a more stable NPFF analog (called (1DMe)Y8Fa(18)) has alsobeen radioiodinated and the binding characterized in spinal cordmembranes (32). The binding was saturable and of high affinity;inhibition of binding with unlabeled NPFF analogs yielded Ki values of0.16 nM and 0.29 nM for (1DMe)Y8Fa and NPFF, respectively, with aBmax=15 fmol/mg protein. No inhibition by various opioid compounds(naloxone, morphine, enkephalins, dynorphins, etc.) or other peptides(NPY, SP, CGRP, for examples) was observed at a concentration of 10 μM,confirming the specificity of NPFF receptors. Interestingly, the relatedmolluscan peptide FMRFa inhibited the binding of [¹²⁵I](1DMe)Y8Fa with aKi=30 nM. The effectiveness of FMRFamide and the C-terminal fragmentNPFF (6-8) at NPFF receptors suggests an important role for the commonC-terminus. Full activity is retained by NPFF (3-8); it has beensuggested that although the C-terminus is important for receptorrecognition, the N-terminus is necessary for formation of ahigh-affinity conformation (33).

[0009] Allard et al. (29) examined the distribution of NPFF bindingsites in rat brain and spinal cord using [¹²⁵I]Y8Fa ([¹²⁵I]YLFQPQRFamide). The highest densities were observed in the external layers of dorsalhorn of spinal cord, several brainstem nuclei, the suprachiasmaticnucleus, restricted nuclei of the thalamus, and the presubiculum of thehippocampus. Lower densities were seen in central gray, reticularformation, ventral tegmental area, lateral and anterior hypothalamus,medial preoptic area, lateral septum, the head of caudate-putamen andcingulate cortex. No binding was observed in cortex, nucleus accumbens,hippocampus (except in presubiculum) or cerebellum. The localization ofNPFF binding sites is in good agreement with the location of the peptideitself, consistent with the binding sites mediating the biologicalactions of NPFF in these tissues (29, 34, 35). Less is known about thesignal transduction pathways activated by NPFF receptors; NPFF was shownto activate adenylyl cyclase in mouse olfactory bulb membranes (36) butno other reports of functional coupling via G proteins have appeared.

[0010] Until now, no direct evidence for NPFF receptor subtypes has beenreported in mammals. Recent physiological data suggest complex(biphasic) effects on nociception and antiopiate activity of NPFF (forreview, see (3, 4)) that could possibly signal the presence of multiplesubtypes. Short term ICV injection of NPFF causes a hyperesthesic effectfollowed by long lasting analgesic effect. Intrathecal NPFF and FMRFaboth produce long-lasting analgesia, but subeffective doses causeddifferent modulatory effects on morphine-induced analgesia (F8Fapotentiated, FMRFa decreased). The analgesic effects of NPFF aresensitive to naloxone, suggesting that NPFF receptors may have distinctpresynaptic (increase release of opioids?) and postsynaptic(anti-opiate?) effects mediated by multiple receptors. Little is knownof the biological effects of A18Famide, which shares its C-terminal 4amino acids with NPFF, but the existence of a family of related peptidesoften is predictive of multiple receptor subtypes.

[0011] No nonpeptide agonists or antagonists of NPFF are available, butseveral useful peptidic analogs have been developed that exhibitincreased agonist stability or antagonist activity. For example,desamino Y8Fa (daY8Fa) can antagonize the behavioral effects of NPFF andrestore morphine-sensitivity (tail-flick analgesia) to morphine-tolerantrats at lower doses, although at higher doses it can act as NPFF agonist(10)(see also (3)). (1DMe)Y8Fa, in which L-Phe¹ is replaced by D-Tyr andthe second peptidic bond is N-methylated, has been shown to inhibitmorphine-induced analgesia (18), and has higher affinity and stabilitythan Y8Fa: (1DMe)Y8Fa was 90% stable after 150 min. incubation with ratspinal cord membranes compared with Y8Fa, which was fully degraded after30 minutes. These analogs may be useful in predicting the effects ofagonist or antagonist drugs that would act at NPFF receptors.

[0012] Despite the numerous studies linking NPFF with analgesia (forreview, see (4)), only recently has NPFF been observed to play a role inanimal models of chronic pain. For example, NPFF has recently been shownto be involved in inflammatory pain (37) and neuropathic pain (38).Importantly, NPFF was shown to attenuate the allodynia associated withneuropathic pain, suggesting that it may be clinically useful intreating this condition. In addition to its potential therapeutic rolesin the treatment of pain and morphine tolerance ((4) and above) NPFF andrelated peptides have a number of other biological activities that maybe therapeutically relevant including effects on feeding (39-41),psychotic behavior (42), nicotine addiction (43), and cardiovascularfunctions (24, 25). The cloning of NPFF receptors will facilitate theelucidation of the roles of NPFF and related peptides in these and otherimportant biological functions.

[0013] Described herein is the isolation and characterization of a newfamily of neuropeptide FF (NPFF) receptors, referred to herein as theNPFF receptors. Cloned NPFF receptors will serve as invaluable tools fordrug design for pathophysiological conditions such as memory loss,affective disorders, schizophrenia, pain, hypertension, locomotorproblems, circadian rhythm disorders, eating/body weight disorders,sexual/reproductive disorders, nasal congestion, diarrhea,gastrointestinal, and cardiovascular disorders.

SUMMARY OF THE INVENTION

[0014] This invention provides an isolated nucleic acid encoding amammalian NPFF receptor.

[0015] This invention provides a nucleic acid encoding a mammalian NPFFreceptor, wherein the nucleic acid (a) hybridizes to a nucleic acidhaving the defined sequence shown in FIG. 1 (Seq. ID No. 1) under lowstringency conditions or a sequence complementary thereto and (b) isfurther characterized by its ability to cause a change in the pH of aculture of CHO cells when a NPFF peptide is added to the culture and theCHO cells express the nucleic acid which hybridized to the nucleic acidhaving the defined sequence or its complement. This invention furtherprovides a nucleic acid encoding a mammalian NPFF receptor, wherein thenucleic acid (a) hybridizes to a nucleic acid having the definedsequence shown in FIG. 4 (Seq. ID No. 3) under low stringency conditionsor a sequence complementary thereto and (b) is further characterized byits ability to cause a change in the pH of a culture of CHO cells when aNPFF peptide is added to the culture and the CHO cells express thenucleic acid which hybridized to the nucleic acid having the definedsequence or its complement. This invention also provides a nucleic acidencoding a mammalian NPFF receptor, wherein the nucleic acid (a)hybridizes to a nucleic acid having the defined sequence shown in FIG. 7(Seq. ID No. 5) under low stringency conditions or a sequencecomplementary thereto and (b) is further characterized by its ability tocause a change in the pH of a culture of CHO cells when a NPFF peptideis added to the culture and the CHO cells express the nucleic acid whichhybridized to the nucleic acid having the defined sequence or itscomplement.

[0016] This invention further provides a nucleic acid encoding amammalian NPFF receptor, wherein the nucleic acid (a) hybridizes to anucleic acid having the defined sequence shown in FIG. 11 (Seq. ID No.7) under low stringency conditions or a sequence complementary theretoand (b) is further characterized by its ability to cause a change in thepH of a culture of CHO cells when a NPFF peptide is added to the cultureand the CHO cells express the nucleic acid which hybridized to thenucleic acid having the defined sequence or its complement.

[0017] This invention also provides a purified mammalian NPFF receptorprotein.

[0018] This invention further provides a vector comprising a nucleicacid encoding a mammalian NPFF receptor, particularly a vector adaptedfor expression of the mammalian NPFF receptor in mammalian ornon-mammalian cells.

[0019] This invention provides a plasmid designated pEXJ-rNPFF1 (ATCCAccession No. 203184). This invention also provides a plasmid designatedpWE15-hNPFF1 (ATCC Accession No. 203183). This invention furtherprovides a plasmid designated pCDNA3.1-hNPFF2b (ATCC Accession No.203255). This invention still further provides a plasmid designatedpcDNA3.1-hNPFF1 (ATCC Accession No. 203605).

[0020] This invention additionally provides a cell comprising a vectorwhich in turn comprises a nucleic acid encoding a mammalian NPFFreceptor as well as a membrane preparation isolated from such a cell.

[0021] Moreover, this invention provides a nucleic acid probe comprisingat least 15 nucleotides, which probe specifically hybridizes with anucleic acid encoding a mammalian NPFF receptor, wherein the probe has aunique sequence corresponding to a sequence present within one of thetwo strands of the nucleic acid encoding the mammalian NPFF1 receptorand contained in plasmid pEXJ-rNPFF1 (ATCC Accession No. 203184),plasmid pWE15-hNPFF1 (ATCC Accession No. 203183), plasmidpCDNA3.1-hNPFF2b (ATCC Accession No. 203255), or plasmid pcDNA3.1-hNPFF1(ATCC Accession No. 203605).

[0022] This invention further provides a nucleic acid probe comprisingat least 15 nucleotides, which probe specifically hybridizes with anucleic acid encoding a mammalian NPFF receptor, wherein the probe has aunique sequence corresponding to a sequence present within (a) thenucleic acid sequence shown in FIG. 1 (Seq. I.D. No. 1) or (b) thereverse complement thereto.

[0023] This invention further provides a nucleic acid probe comprisingat least 15 nucleotides, which probe specifically hybridizes with anucleic acid encoding a mammalian NPFF receptor, wherein the probe has aunique sequence corresponding to a sequence present within (a) thenucleic acid sequence shown in FIG. 4 (Seq. I.D. No. 3) or (b) thereverse complement thereto.

[0024] This invention further provides a nucleic acid probe comprisingat least 15 nucleotides, which probe specifically hybridizes with anucleic acid encoding a mammalian NPFF receptor, wherein the probe has aunique sequence corresponding to a sequence present within (a) thenucleic acid sequence shown in FIG. 7 (Seq. I.D. No. 5) or (b) thereverse complement thereto.

[0025] This invention further provides a nucleic acid probe comprisingat least 15 nucleotides, which probe specifically hybridizes with anucleic acid encoding a mammalian NPFF receptor, wherein the probe has aunique sequence corresponding to a sequence present within (a) thenucleic acid sequence shown in FIG. 11 (Seq. I.D. No. 7) or (b) thereverse complement thereto.

[0026] This invention still further provides an antisenseoligonucleotide having a sequence capable of specifically hybridizing toRNA encoding the mammalian NPFF receptor, so as to prevent translationof the RNA. This invention also provides an antisense oligonucleotidehaving a sequence capable of specifically hybridizing to genomic DNAencoding a mammalian NPFF receptor, so as to prevent transcriptionthereof.

[0027] This invention further provides an antibody capable of binding toa mammalian NPFF receptor. This invention also provides an agent capableof competitively inhibiting the binding of the antibody to a mammalianNPFF receptor.

[0028] In addition, this invention provides a pharmaceutical compositioncomprising (a) an amount of the oligonucleotide described above capableof passing through a cell membrane and effective to reduce expression ofa mammalian NPFF receptor and (b) a pharmaceutically acceptable carriercapable of passing through the cell membrane.

[0029] This invention also provides a transgenic, nonhuman mammalexpressing DNA encoding a mammalian NPFF receptor. This invention alsoprovides a transgenic, nonhuman mammal comprising a homologousrecombination knockout of the native mammalian NPFF receptor. Thisinvention further provides a transgenic, nonhuman mammal whose genomecomprises antisense DNA complementary to the DNA encoding a mammalianNPFF receptor so placed within the genome as to be transcribed intoantisense mRNA which is complementary to mRNA encoding the mammalianNPFF receptor and which hybridizes to mRNA encoding the mammalian NPFFreceptor, thereby reducing its translation.

[0030] This invention provides a process for identifying a chemicalcompound which specifically binds to a mammalian NPFF receptor whichcomprises contacting cells containing DNA encoding and expressing ontheir cell surface the mammalian NPFF receptor, wherein such cells donot normally express the mammalian NPFF receptor, with the compoundunder conditions suitable for binding, and detecting specific binding ofthe chemical compound to the mammalian NPFF receptor.

[0031] This invention further provides a process for identifying achemical compound which specifically binds to a mammalian NPFF receptorwhich comprises contacting a membrane preparation from cells transfectedwith DNA encoding and expressing on their cell surface the mammalianNPFF receptor, wherein such cells do not normally express the mammalianNPFF receptor, with the compound under conditions suitable for binding,and detecting specific binding of the chemical compound to the mammalianNPFF receptor.

[0032] This invention provides a process involving competitive bindingfor identifying a chemical compound which specifically binds to amammalian NPFF receptor which comprises separately contacting cellsexpressing on their cell surface the mammalian NPFF receptor, whereinsuch cells do not normally express the mammalian NPFF receptor, withboth the chemical compound and a second chemical compound known to bindto the receptor, and with only the second chemical compound, underconditions suitable for binding of both compounds, and detectingspecific binding of the chemical compound to the mammalian NPFFreceptor, a decrease in the binding of the second chemical compound tothe mammalian NPFF receptor in the presence of the chemical compoundindicating that the chemical compound binds to the mammalian NPFFreceptor.

[0033] This invention further provides a process involving competitivebinding for identifying a chemical compound which specifically binds toa mammalian NPFF receptor which comprises separately contacting amembrane fraction from cells expressing on their cell surface themammalian NPFF receptor, wherein such cells do not normally express themammalian NPFF receptor, with both the chemical compound and a secondchemical compound known to bind to the receptor, and with only thesecond chemical compound, under conditions suitable for binding of bothcompounds, and detecting specific binding of the chemical compound tothe mammalian NPFF receptor, a decrease in the binding of the secondchemical compound to the mammalian NPFF receptor in the presence of thechemical compound indicating that the chemical compound binds to themammalian NPFF receptor.

[0034] This invention provides a method of screening a plurality ofchemical compounds not known to bind to a mammalian NPFF receptor toidentify a compound which specifically binds to the mammalian NPFFreceptor, which comprises (a) contacting cells transfected with andexpressing DNA encoding the mammalian NPFF receptor with a compoundknown to bind specifically to the mammalian NPFF receptor; (b)contacting the preparation of step (a) with the plurality of compoundsnot known to bind specifically to the mammalian NPFF receptor, underconditions permitting binding of compounds known to bind to themammalian NPFF receptor; (c) determining whether the binding of thecompound known to bind to the mammalian NPFF receptor is reduced in thepresence of any compound within the plurality of compounds, relative tothe binding of the compound in the absence of the plurality ofcompounds; and if so (d) separately determining the binding to themammalian NPFF receptor of compounds included in the plurality ofcompounds, so as to thereby identify the compound which specificallybinds to the mammalian NPFF receptor.

[0035] This invention also provides a method of screening a plurality ofchemical compounds not known to bind to a mammalian NPFF receptor toidentify a compound which specifically binds to the mammalian NPFFreceptor, which comprises (a) contacting a membrane preparation fromcells transfected with and expressing DNA encoding a mammalian NPFFreceptor with a compound known to bind to the mammalian NPFF receptor;(b) determining whether the binding of a compound known to bind to themammalian NPFF receptor is reduced in the presence of any compoundwithin the plurality of compounds, relative to the binding of thecompound in the absence of the plurality of compounds; and if so (c)separately determining the binding to the mammalian NPFF receptor ofcompounds included in the plurality of compounds, so as to therebyidentify the compound which specifically binds to the mammalian NPFFreceptor.

[0036] Still further, this invention provides a method of detectingexpression of a mammalian NPFF receptor by detecting the presence ofmRNA coding for the mammalian NPFF receptor which comprises obtainingtotal mRNA from the cell and contacting the mRNA so obtained with anucleic acid probe under hybridizing conditions, detecting the presenceof mRNA hybridizing to the probe, and thereby detecting the expressionof the mammalian NPFF receptor by the cell.

[0037] This invention provides a method of detecting the presence of amammalian NPFF receptor on the surface of a cell which comprisescontacting the cell with an antibody under conditions permitting bindingof the antibody to the receptor, detecting the presence of the antibodybound to the cell, and thereby detecting the presence of the mammalianNPFF receptor on the surface of the cell.

[0038] This invention provides a method of determining the physiologicaleffects of varying levels of activity of mammalian NPFF receptors whichcomprises producing a transgenic, nonhuman mammal whose levels ofmammalian NPFF receptor activity are varied by use of an induciblepromoter which regulates mammalian NPFF receptor expression.

[0039] This invention also provides a method of determining thephysiological effects of varying levels of activity of mammalian NPFFreceptors which comprises producing a panel of transgenic, nonhumanmammals each expressing a different amount of mammalian NPFF receptor.

[0040] This invention further provides a method for identifying anantagonist capable of alleviating an abnormality wherein the abnormalityis alleviated by decreasing the activity of a mammalian NPFF receptorcomprising administering a compound to a transgenic, nonhuman mammal asdescribed above and determining whether the compound alleviates thephysical and behavioral abnormalities displayed by the transgenic,nonhuman mammal as a result of overactivity of a mammalian NPFFreceptor, the alleviation of the abnormality identifying the compound asan antagonist. This invention also provides an antagonist identified bythis method. This invention still further provides a pharmaceuticalcomposition comprising an antagonist identified by this method and apharmaceutically acceptable carrier.

[0041] This invention additionally provides a method of treating anabnormality in a subject wherein the abnormality is alleviated bydecreasing the activity of a mammalian NPFF receptor which comprisesadministering to the subject an effective amount of the precedingpharmaceutical composition containing a mammalian NPFF receptorantagonist, thereby treating the abnormality.

[0042] This invention also provides a method for identifying an agonistcapable of alleviating an abnormality in a subject wherein theabnormality is alleviated by increasing the activity of a mammalian NPFFreceptor comprising administering a compound to a transgenic, nonhumanmammal, and determining whether the compound alleviates the physical andbehavioral abnormalities displayed by the transgenic, nonhuman mammal,the alleviation of the abnormality identifying the compound as anagonist. This invention also provides an agonist identified by thismethod. This invention further provides a pharmaceutical compositioncomprising an agonist identified by this method and a pharmaceuticallyacceptable carrier. This invention provides a method of treating anabnormality in a subject wherein the abnormality is alleviated byincreasing the activity of a mammalian NPFF receptor which comprisesadministering to the subject an effective amount of the precedingpharmaceutical composition containing a mammalian NPFF receptor agonist,thereby treating the abnormality

[0043] This invention provides a method for diagnosing a predispositionto a disorder associated with the activity of a specific mammalianallele which comprises: (a) obtaining DNA of subjects suffering from thedisorder; (b) performing a restriction digest of the DNA with a panel ofrestriction enzymes; (c) electrophoretically separating the resultingDNA fragments on a sizing gel; (d) contacting the resulting gel with anucleic acid probe capable of specifically hybridizing with a uniquesequence included within the sequence of a nucleic acid moleculeencoding a mammalian NPFF receptor and labeled with a detectable marker;(e) detecting labeled bands which have hybridized to the DNA encoding amammalian NPFF receptor labeled with a detectable marker to create aunique band pattern specific to the DNA of subjects suffering from thedisorder; (f) preparing DNA obtained for diagnosis by steps (a)-(e); and(g) comparing the unique band pattern specific to the DNA of subjectssuffering from the disorder from step (e) and the DNA obtained fordiagnosis from step (f) to determine whether the patterns are the sameor different and to diagnose thereby predisposition to the disorder ifthe patterns are the same.

[0044] This invention provides a method of preparing a purifiedmammalian NPFF receptor which comprises: (a)culturing cells whichexpress the mammalian NPFF receptor; (b) recovering the mammalian NPFFreceptor from the cells; and (c) purifying the mammalian NPFF receptorso recovered.

[0045] This invention provides a method of preparing a purifiedmammalian NPFF receptor which comprises: (a)inserting a nucleic acidencoding the mammalian NPFF receptor into a suitable vector; (b)introducing the resulting vector into a suitable host cell; (c) placingthe resulting cell in suitable condition permitting the production ofthe mammalian NPFF receptor; (d) recovering the mammalian NPFF receptorproduced by the resulting cell; and (e) isolating and/or purifying themammalian NPFF receptor so recovered.

[0046] This invention provides a process for determining whether achemical compound is a mammalian NPFF receptor agonist which comprisescontacting cells transfected with and expressing DNA encoding themammalian NPFF receptor with the compound under conditions permittingthe activation of the mammalian NPFF receptor, and detecting an increasein mammalian NPFF receptor activity, so as to thereby determine whetherthe compound is a mammalian NPFF receptor agonist. This invention alsoprovides a pharmaceutical composition which comprises an amount of amammalian NPFF receptor agonist determined by this process effective toincrease activity of a mammalian NPFF receptor and a pharmaceuticallyacceptable carrier.

[0047] This invention provides a process for determining whether achemical compound is a mammalian NPFF receptor antagonist whichcomprises contacting cells transfected with and expressing DNA encodingthe mammalian NPFF receptor with the compound in the presence of a knownmammalian NPFF receptor agonist, under conditions permitting theactivation of the mammalian NPFF receptor, and detecting a decrease inmammalian NPFF receptor activity, so as to thereby determine whether thecompound is a mammalian NPFF receptor antagonist. This invention alsoprovides a pharmaceutical composition which comprises an amount of amammalian NPFF receptor antagonist determined by this process effectiveto reduce activity of a mammalian NPFF receptor and a pharmaceuticallyacceptable carrier.

[0048] This invention provides a process for determining whether achemical compound specifically binds to and activates a mammalian NPFFreceptor, which comprises contacting cells producing a second messengerresponse and expressing on their cell surface the mammalian NPFFreceptor, wherein such cells do not normally express the mammalian NPFFreceptor, with the chemical compound under conditions suitable foractivation of the mammalian NPFF receptor, and measuring the secondmessenger response in the presence and in the absence of the chemicalcompound, a change in the second messenger response in the presence ofthe chemical compound indicating that the compound activates themammalian NPFF receptor. This invention also provides a compounddetermined by this process. This invention further provides apharmaceutical composition which comprises an amount of the compound (aNPFF receptor agonist) determined by this process effective to increaseactivity of a mammalian NPFF receptor and a pharmaceutically acceptablecarrier.

[0049] This invention provides a process for determining whether achemical compound specifically binds to and inhibits activation of amammalian NPFF receptor, which comprises separately contacting cellsproducing a second messenger response and expressing on their cellsurface the mammalian NPFF receptor, wherein such cells do not normallyexpress the mammalian NPFF receptor, with both the chemical compound anda second chemical compound known to activate the mammalian NPFFreceptor, and with only the second chemical compound, under conditionssuitable for activation of the mammalian NPFF receptor, and measuringthe second messenger response in the presence of only the secondchemical compound and in the presence of both the second chemicalcompound and the chemical compound, a smaller change in the secondmessenger response in the presence of both the chemical compound and thesecond chemical compound than in the presence of only the secondchemical compound indicating that the chemical compound inhibitsactivation of the mammalian NPFF receptor. This invention also providesa compound determined by this process. This invention further provides apharmaceutical composition which comprises an amount of the compound (amammalian NPFF receptor antagonist) determined by this effective toreduce activity of a mammalian NPFF receptor and a pharmaceuticallyacceptable carrier.

[0050] This invention provides a method of screening a plurality ofchemical compounds not known to activate a mammalian NPFF receptor toidentify a compound which activates the mammalian NPFF receptor whichcomprises: (a) contacting cells transfected with and expressing themammalian NPFF receptor with the plurality of compounds not known toactivate the mammalian NPFF receptor, under conditions permittingactivation of the mammalian NPFF receptor; (b) determining whether theactivity of the mammalian NPFF receptor is increased in the presence ofthe compounds; and if so (c) separately determining whether theactivation of the mammalian NPFF receptor is increased by each compoundincluded in the plurality of compounds, so as to thereby identify thecompound which activates the mammalian NPFF receptor. This inventionalso provides a compound identified by this method. This inventionfurther provides a pharmaceutical composition which comprises an amountof the compound (a mammalian NPFF receptor agonist) identified by thismethod effective to increase activity of a mammalian NPFF receptor and apharmceutically acceptable carrier.

[0051] This invention provides a method of screening a plurality ofchemical compounds not known to inhibit the activation of a mammalianNPFF receptor to identify a compound which inhibits the activation ofthe mammalian NPFF receptor, which comprises: (a) contacting cellstransfected with and expressing the mammalian NPFF receptor with theplurality of compounds in the presence of a known mammalian NPFFreceptor agonist, under conditions permitting activation of themammalian NPFF receptor; (b) determining whether the activation of themammalian NPFF receptor is reduced in the presence of the plurality ofcompounds, relative to the activation of the mammalian NPFF receptor inthe absence of the plurality of compounds; and if so (c) separatelydetermining the inhibition of activation of the mammalian NPFF receptorfor each compound included in the plurality of compounds, so as tothereby identify the compound which inhibits the activation of themammalian NPFF receptor. This invention also provides a compoundidentified by this method. This invention further provides apharmaceutical composition which comprises an amount of the compound (amammalian NPFF receptor antagonist) identified by this process effectiveto decrease activity of a mammalian NPFF receptor and a pharmaceuticallyacceptable carrier.

[0052] This invention provides a method of treating an abnormality in asubject wherein the abnormality is alleviated by increasing the activityof a mammalian NPFF receptor which comprises administering to thesubject an amount of a compound which is a mammalian NPFF receptoragonist effective to treat the abnormality.

[0053] This invention provides a method of treating an abnormality in asubject wherein the abnormality is alleviated by decreasing the activityof a mammalian NPFF receptor which comprises administering to thesubject an amount of a compound which is a mammalian NPFF receptorantagonist effective to treat the abnormality.

[0054] This invention provides a process for making a composition ofmatter which specifically binds to a mammalian NPFF receptor whichcomprises identifying a chemical compound using any of the processesdescribed herein for identifying a compound which binds to and/oractivates or inhibits activation of a mammalian NPFF receptor and thensynthesizing the chemical compound or a novel structural and functionalanalog or homolog thereof. This invention further provides a process forpreparing a pharmaceutical composition which comprises admixing apharmaceutically acceptable carrier and a pharmaceutically acceptableamount of a chemical compound identified by any of the processesdescribed herein for identifying a compound which binds to and/oractivates or inhibits activation of a mammalian NPFF receptor or a novelstructural and functional analog or homolog thereof.

BRIEF DESCRIPTION OF THE FIGURES

[0055]FIG. 1

[0056] Nucleotide sequence encoding a rat neuropeptide FF receptor(NPFF1) (Seq. I.D. No. 1). In addition, partial 5′ and 3′ untranslatedsequences are shown. In FIG. 1, two start (ATG) codons (at positions73-75 and 148-150) and the stop (TAG) codon (at positions 1369-1371) areunderlined.

[0057]FIG. 2

[0058] Deduced amino acid sequence (Seq. I.D. No. 2) of the ratneuropeptide FF receptor (NPFF1) encoded by the nucleotide sequenceshown FIGS. 1 (Seq. I.D. No. 1).

[0059]FIG. 3

[0060] Deduced amino acid sequence for rat NPFF1 (SEQ. I.D. No. 2).Seven solid lines designated I-VII located above portions of thesequence indicate the seven putative transmembrane (TM) spanningregions.

[0061]FIG. 4

[0062] Partial coding sequence of human neuropeptide FF receptor (NPFF1)(SEQ. I.D. No. 3).

[0063]FIG. 5

[0064] Partial deduced amino acid sequence of the human neuropeptide FF(NPFF1) receptor (SEQ. I.D. No. 4) encoded by the partial nucleotidesequence of FIG. 3.

[0065]FIG. 6

[0066] Partial amino acid alignment of rat and human NPFF1. Verticallines represent identical residues and dots represent similar residues.

[0067]FIG. 7

[0068] Nucleotide sequence of hNPFF2b (SEQ. I.D. No. 5). The initiatingmethionine and the stop codon are underlined.

[0069]FIG. 8

[0070] Deduced amino acid sequence of human NPFF2b (hNPFF2) (Seq. I.D.No. 6) encoded by the nucleotide sequence shown in FIG. 7.

[0071]FIG. 9

[0072] Deduced amino acid sequence for human hNPFF2 (SEQ. I.D. No. 6),with potential transmembrane domains underlined.

[0073]FIG. 10

[0074] Amino acid alignment of rat NPFF1 and human NPFF2. Vertical linesrepresent identical residues and dots represent similar residues.

[0075] Figure Legends

[0076]FIG. 11

[0077] Nucleotide sequence of a human neuropeptide FF receptor (NPFF1)(Seq. I.D. No. 7). The initiating methionine (at positions 1-3) and thestop codon (at positions 1291-1293) are underlined.

[0078]FIG. 12

[0079] Deduced amino acid sequence of the human neuropeptide FF receptor(NPFF1) (Seq. I.D. No. 8).

[0080]FIG. 13

[0081] Deduced amino acid sequence for human NPFF1 (Seq. I.D. No. 8).Seven solid lines designated I-VII indicate the seven putativetransmembrane (TM) spanning regions.

[0082]FIG. 14

[0083] Amino acid alignment of the human NPFF1 and human NPFF2receptors. Vertical lines represent identical residues and dotsrepresent similar residues.

[0084]FIG. 15A-15C

[0085] Electrophysiological responses to NPFF and related peptides fromvoltage clamped oocytes expressing NPFF1 and chimeric G-protein.

[0086] FIGS. 16A-16C

[0087] Electrophysiological responses in voltage-clamped oocytesexpressing NPFF2 mRNA.

[0088]FIG. 16A: Oocyte injected with NPFF2 mRNA (from ligation PCR)generates an inward current in response to NPFF at 1 μM.

[0089]FIG. 16B: In a different oocyte, no response is observed whenchallenged with a mixture of galanin, NPY, orexin A and neurokinin A,each at 1 μM. A subsequent application of NPFF elicits a response.

[0090]FIG. 16C: Oocyte injected with NPFF2 mRNA (from BO89) generates aninward current in response to NPFF at 1 μM. Oocytes were clamped at aholding potential of −80 mV.

[0091]FIGS. 17A and 17B

[0092] Microphysiometric response of CHO cells transiently transfectedwith either NPFF1 (SN2) alone or NPFF1 accompanied by Gq/Gz.

[0093]FIG. 17A: Cells expressing either NPFF1 alone or NPFF1+Gq/Gzproduced a dose-dependent response to NPFF with an EC50 value of 19.3 nMand 27.7 nM respectively. Mock control cells transfected with emptyvector produced little if any response to NPFF even at the highestconcentrations used.

[0094]FIG. 17B: Cells expressing NPFF1 alone produced a dose-dependentresponse to A-18-F-amide with an EC50 value of 150 nM. In both FIGS. 17Aand 17B control cells mock transfected with empty vector produced littleif any response to drug even at the highest concentrations used.Responses are reported as percentage increase in the acidification rateas observed just prior to drug challenge (immediate prior basal rate).

[0095]FIGS. 18A and 18B

[0096] NPFF stimulation of Inositol phosphate release in NPFF-1transfected Cos-7 cells.

[0097]FIG. 18A: Cos-7 cells were transiently transfected with NPFF-1receptor cDNA.

[0098]FIG. 18B: Cos-7 cells were transiently co-transfected with cDNAsfor the NPFF-1 receptor and the Gq/Gz chimera. The accumulation of totalinositol phosphate release was measured by prelabelling cells with[³H]myoinositol (2 μCi/ml) overnight. Cells were washed to removeunincorporated radioactivity and resuspended in medium containing 10 mMLiCl. [³H]myoinositol labeled cells were incubated with appropriatedrugs for 1 hr at 37° C. The reaction was stopped by addition of 5% TCAand IPs were isolated by ion exchange chromatography (Berridge et al.,1982). Columns were washed with water and total [³H] inositol phosphateswere then eluted with 1M ammonium formate/0.1 M formic acid.Radioactivity in the final fraction was measured by liquid scintillationspectroscopy. Cells were either treated with vehicle (water, control) orcholera toxin (CTX; 1 μg/ml) or pertussis toxin (PTX, 100 ng/ml)overnight. Data are from one experiment representative of at least oneother.

[0099]FIG. 19

[0100] RT-PCR was performed as described on a panel of mRNA extractedfrom rat tissue as indicated at the bottom of the gel. Afteramplification, PCR reactions were size fractionated on 10%polyacrylamide gels, and stained with SYBR Green I. Images were analyzedusing a Molecular Dynamics Storm 860 workstation. The amplified bandcorresponding to NPFF1 (490 base pairs) is indicated (arrow). RT-PCRindicates a broad distribution of mRNA encoding NPFF1 with highestconcentrations found in nervous system structures.

[0101]FIG. 20

[0102] Autoradiograph demonstrating hybridization of radiolabeled ratNPFF1 probe to RNA extracted from rat tissue in a solutionhybridization/nuclease protection assay using ³²P labeled riboprobe. 2μg of RNA was used in each assay. The single band (arrow) representsmRNA coding for the NPFF1 receptors extracted from the indicated tissue.Highest levels of mRNA coding for NPFF1 are found in:

[0103] hypothalamus and pituitary gland. The smaller bands representingNPFF1 mRNA from the pituitary, adrenal gland, and ovary (double arrow)may indicate a splice variant present in this tissue. Integrity of RNAwas assessed using hybridization to mRNA coding for GAPDH (not shown).

[0104]FIG. 21

[0105] RT-PCR was performed as described on a panel of mRNA extractedfrom tissue as indicated at the bottom of the gel. After amplification,PCR reactions were size fractionated on 10% polyacrylamide gels, andstained with SYBR Green I. Images were analyzed using a MolecularDynamics Storm 860 workstation. The amplified band corresponding toNPFF2 receptors (approximately 325 base pairs) is indicated (arrow).RT-PCR indicates a broad distribution of mRNA encoding NPFF2 receptors.The only tissue containing mRNA coding for NPFF2 receptors were HeLacells and Jurkat cells.

DETAILED DESCRIPTION OF THE INVENTION

[0106] Throughout this application, the following standard abbreviationsare used to indicate specific nucleotide bases: A = adenine G = guanineC = cytosine T = thymine U = uracil M = adenine or cytosine R = adenineor guanine W = adenine, thymine, or uracil S = cytosine or guanine Y =cytosine, thymine, or uracil K = guanine, thymine, or uracil V =adenine, cytosine, or guanine (not thymine or uracil H = adenine,cytosine, thymine, or uracil (not guanine) D = adenine, guanine,thymine, or uracil (not cytosine) B = cytosine, guanine, thymine, oruracil (not adenine) N = adenine, cytosine, guanine, thymine, or uracil(or other modified base such as inosine) I = inosine

[0107] Furthermore, the term “agonist” is used throughout thisapplication to indicate any peptide or non-peptidyl compound whichincreases the activity of any of the polypeptides of the subjectinvention. The term “antagonist” is used throughout this application toindicate any peptide or non-peptidyl compound which decreases theactivity of any of the polypeptides of the subject invention.

[0108] The activity of a G-protein coupled receptor such as thepolypeptides disclosed herein may be measured using any of a variety offunctional assays in which activation of the receptor in questionresults in an observable change in the level of some second messengersystem, including, but not limited to, adenylate cyclase, calciummobilization, arachidonic acid release, ion channel activity, inositolphospholipid hydrolysis or guanylyl cyclase. Heterologous expressionsystems utilizing appropriate host cells to express the nucleic acid ofthe subject invention are used to obtain the desired second messengercoupling. Receptor activity may also be assayed in an oocyte expressionsystem.

[0109] It is possible that the mammalian NPFF receptor genes containintrons and furthermore, the possibility exists that additional intronscould exist in coding or non-coding regions. In addition, splicedform(s) of mRNA may encode additional amino acids either upstream of thecurrently defined starting methionine or within the coding region.Further, the existence and use of alternative exons is possible, wherebythe mRNA may encode different amino acids within the region comprisingthe exon. In addition, single amino acid substitutions may arise via themechanism of RNA editing such that the amino acid sequence of theexpressed protein is different than that encoded by the original gene.(Burns et al., 1996; Chu et al., 1996). Such variants may exhibitpharmacologic properties differing from the polypeptide encoded by theoriginal gene.

[0110] This invention provides splice variants of the mammalian NPFFreceptors disclosed herein. This invention further provides foralternate translation initiation sites and alternately spliced or editedvariants of nucleic acids encoding the mammalian NPFF receptors of thisinvention.

[0111] The nucleic acids of the subject invention also include nucleicacid analogs of the rat and human NPFF receptor genes, wherein the ratNPFF1 receptor gene comprises the nucleic acid sequence shown in FIG. 1or contained in plasmid pEXJ-rNPFF1 (ATCC Accession No. 203184); thehuman NPFF1 receptor gene comprises the nucleic acid shown in FIG. 4 andcontained in plasmid pWE15-hNPFF1 (ATCC Accession No. 203183); the humanNPFF2 receptor gene comprises the nucleic acid shown in FIG. 7 andcontained in plasmid pCDNA3.1-hNPFF2b (ATCC Accession No. 203255); orthe human NPFF1 receptor gene comprises the nucleic acid shown in FIG.11 and contained in plasmid pcDNA3.1-hNPFF1 (ATCC Accession No. 203605).Nucleic acid analogs of the rat and human NPFF receptor genes differfrom the rat and human NPFF receptor genes described herein in terms ofthe identity or location of one or more nucleic acid bases (deletionanalogs containing less than all of the nucleic acid bases shown inFIGS. 1, 4, 7 or 11 or contained in plasmids pEXJ-rNPFF1, pWE15-hNPFF1,pCDNA3.1-hNPFF2b or pcDNA3.1-hNPFF1 respectively, substitution analogswherein one or more nucleic acid bases shown in FIGS. 1, 4, 7 or 11 orcontained in plasmids pEXJ-rNPFF1, pWE15-hNPFF1, pCDNA3.1-hNPFF2b orpcDNA3.1-hNPFF1, respectively, are replaced by other nucleic acid bases,and addition analogs, wherein one or more nucleic acid bases are addedto a terminal or medial portion of the nucleic acid sequence) and whichencode proteins which share some or all of the properties of theproteins encoded by the nucleic acid sequences shown in FIGS. 1, 4, 7 or11 or contained in plasmids pEXJ-rNPFF1, pWE15-hNPFF1, pCDNA3.1-hNPFF2b,or pcDNA3.1-hNPFF1, respectively. In one embodiment of the presentinvention, the nucleic acid analog encodes a protein which has an aminoacid sequence identical to that shown in FIG. 2, 5 8 or 12 or encoded bythe nucleic acid sequence contained in plasmids pEXJ-rNPFF1,pWE15-hNPFF1, pCDNA3.1-hNPFF2b or pcDNA3.1-hNPFF1, respectively. Inanother embodiment, the nucleic acid analog encodes a protein having anamino acid sequence which differs from the amino acid sequences shown inFIG. 2, 5, 8 or 12 or encoded by the nucleic acid contained in plasmidspEXJ-rNPFF1, pWE15-hNPFF1, pCDNA3.1-hNPFF2b or pcDNA3.1-hNPFF1respectively. In a further embodiment, the protein encoded by thenucleic acid analog has a function which is the same as the function ofthe receptor proteins having the amino acid sequence shown in FIG. 2, 5,8 or 12. In another embodiment, the function of the protein encoded bythe nucleic acid analog differs from the function of the receptorprotein having the amino acid sequence shown in FIG. 2, 5, 8 or 12. Inanother embodiment, the variation in the nucleic acid sequence occurswithin the transmembrane (TM) region of the protein. In a furtherembodiment, the variation in the nucleic acid sequence occurs outside ofthe TM region.

[0112] This invention provides the above-described isolated nucleicacid, wherein the nucleic acid is DNA. In an embodiment, the DNA iscDNA. In another embodiment, the DNA is genomic DNA. In still anotherembodiment, the nucleic acid is RNA. Methods for production andmanipulation of nucleic acid molecules are well known in the art.

[0113] This invention further provides nucleic acid which is degeneratewith respect to the DNA encoding any of the polypeptides describedherein. In an embodiment, the nucleic acid comprises a nucleotidesequence which is degenerate with respect to the nucletide sequenceshown in FIGS. 1 (SEQ I.D. No. 1), 4 (SEQ I.D. No. 3), 7 (SEQ I.D. No.5) or 11 (SEQ I.D. No. 7) or the nucleotide sequence contained in theplasmids pEXJ-rNPFF1, pWE15-hNPFF1, pCDNA3.1-hNPFF2b or pcDNA3.1-hNPFF1,respectively, that is, a nucleotide sequence which is translated intothe same amino acid sequence.

[0114] This invention also encompasses DNAs and cDNAs which encode aminoacid sequences which differ from those of the polypeptides of thisinvention, but which should not produce phenotypic changes. Alternately,this invention also encompasses DNAs, cDNAs, and RNAs which hybridize tothe DNA, cDNA, and RNA of the subject invention. Hybridization methodsare well known to those of skill in the art.

[0115] The nucleic acids of the subject invention also include nucleicacid molecules coding for polypeptide analogs, fragments or derivativesof antigenic polypeptides which differ from naturally-occurring forms interms of the identity or location of one or more amino acid residues(deletion analogs containing less than all of the residues specified forthe protein, substitution analogs wherein one or more residues specifiedare replaced by other residues and addition analogs wherein one or moreamino acid residues is added to a terminal or medial portion of thepolypeptides) and which share some or all properties ofnaturally-occurring forms. These molecules include: the incorporation ofcodons “preferred” for expression by selected non-mammalian hosts; theprovision of sites for cleavage by restriction endonuclease enzymes; andthe provision of additional initial, terminal or intermediate DNAsequences that facilitate construction of readily expressed vectors Thecreation of polypeptide analogs is well known to those of skill in theart (R. F. Spurney et al. (1997); Fong, T. M. et al. (1995); Underwood,D. J. et al. (1994); Graziano, M. P. et al. (1996); Guam X. M. et al.(1995)).

[0116] The modified polypeptides of this invention may be transfectedinto cells either transiently or stably using methods well-known in theart, examples of which are disclosed herein. This invention alsoprovides for binding assays using the modified polypeptides, in whichthe polypeptide is expressed either transiently or in stable cell lines.This invention further provides a compound identified using a modifiedpolypeptide in a binding assay such as the binding assays describedherein.

[0117] The nucleic acids described and claimed herein are useful for theinformation which they provide concerning the amino acid sequence of thepolypeptide and as products for the large scale synthesis of thepolypeptides by a variety of recombinant techniques. The nucleic acidmolecule is useful for generating new cloning and expression vectors,transformed and transfected prokaryotic and eukaryotic host cells, andnew and useful methods for cultured growth of such host cells capable ofexpression of the polypeptide and related products.

[0118] This invention provides an isolated nucleic acid encoding amammalian NPFF receptor. In one embodiment, the nucleic acid is DNA. Inanother embodiment, the DNA is cDNA. In another embodiment, the DNA isgenomic DNA. In another embodiment, the nucleic acid is RNA.

[0119] In one embodiment, the mammalian NPFF receptor is a NPFF1receptor. In a further embodiment, the mammalian NPFF1 receptor is a ratNPFF1 receptor. In another embodiment, the mammalian NPFF1 receptor is ahuman NPFF1 receptor. In a further embodiment, the mammalian NPFFreceptor is a NPFF2 receptor. In one embodiment, the mammalian NPFF2receptor is a human NPFF2 receptor.

[0120] This invention also provides an isolated nucleic acid encodingspecies homologs of the NPFF receptors encoded by the nucleic acidsequence shown in FIGS. 1 (Seq. I.D. No. 1), 4 (Seq. I.D. No. 3), 7(Seq. I.D. No. 5), or 11 (Seq. I.D. No. 7) or encoded by the plasmidpEXJ-rNPFF1, pWE15-hNPFF1, pCDNA3.1-hNPFF2b or pcDNA3.1-hNPFF1,respectively. In one embodiment, the nucleic acid encodes a mammalianNPFF receptor homolog which has substantially the same amino acidsequence as does the NPFF receptor encoded by the plasmid pEXJ-rNPFF1,pWE15-hNPFF1, pCDNA3.1-hNPFF2b or pcDNA3.1-hNPFF1. In anotherembodiment, the nucleic acid encodes a mammalian NPFF receptor homologwhich has above 65% amino acid identity to the NPFF receptor encoded bythe plasmid pEXJ-rNPFF1, pWE15-hNPFF1, pCDNA3.1-hNPFF2b. orpcDNA3.1-hNPFF1; preferably above 75% amino acid identity to the NPFFreceptor encoded by the plasmid pEXJ-rNPFF1, pWE15-hNPFF1,pCDNA3.1-hNPFF2b, or pcDNA3.1-hNPFF1; more preferably above 85% aminoacid identity to the NPFF receptor encoded by the plasmid pEXJ-rNPFF1,pWE15-hNPFF1, pCDNA3.1-hNPFF2b, or pcDNA3.1-hNPFF1; most preferablyabove 95% amino acid identity to the NPFF receptor encoded by theplasmid pEXJ-rNPFF1, PWE15-hNPFF1, pCDNA3.1-hNPFF2b, or pcDNA3.1-hNPFF1.In another embodiment, the mammalian NPFF receptor homolog has above 70%nucleic acid identity to the NPFF receptor gene contained in plasmidpEXJ-rNPFF1, pWE15-hNPFF1, pCDNA3.1-hNPFF2b, or pcDNA3.1-hNPFF1;preferably above 80% nucleic acid identity to the NPFF receptor genecontained in the plasmid pEXJ-rNPFF1, pWE15-hNPFF1, pCDNA3.i-hNPFF2b, orpcDNA3.1-hNPFF1; more preferably above 90% nucleic acid identity to theNPFF receptor gene contained in the plasmid pEXJ-rNPFF1, pWE15-hNPFF1,pCDNA3.1-hNPFF2b, or pcDNA3.1-hNPFF1. Examples of methods for isolatingand purifying species homologs are described elsewhere (e.g., U.S. Pat.No. 5,602,024, WO94/14957, WO97/26853, WO98/15570).

[0121] In separate embodiments of the present invention, the nucleicacid encodes a NPFF receptor which has an amino acid sequence identicalto that encoded by the plasmid pEXJ-rNPFF1, pWE15-hNPFF1,pCDNA3.1-hNPFF2b, or pcDNA3.1-hNPFF1. In further embodiments, the NPFFreceptor has a sequence substantially the same as the amino acidsequence shown in FIG. 2 (Seq. I.D. No. 2), FIG. 5 (Seq. I.D. No. 4),FIG. 8 (Seq. I.D. No. 6) or FIG. 12 (Seq. I.D. No. 8). In otherembodiments, the NPFF receptor has an amino acid sequence identical tothe amino acid sequence shown in FIG. 2 (Seq. I.D. No. 2), FIG. 5 (Seq.I.D. No. 4), FIG. 8 (Seq. I.D. No. 6) or FIG. 12 (Seq. I.D. No. 8).

[0122] This invention provides an isolated nucleic acid encoding amodified mammalian NPFF receptor, which differs from a mammalian NPFFreceptor by having an amino acid(s) deletion, replacement, or additionin the third intracellular domain.

[0123] This invention provides a nucleic acid encoding a mammalian NPFFreceptor, wherein the nucleic acid (a) hybridizes to a nucleic acidhaving the defined sequence shown in FIG. 1 (Seq. I.D. No. 1) under lowstringency conditions or a sequence complementary thereto and (b) isfurther characterized by its ability to cause a change in the pH of aculture of CHO cells when a NPFF peptide is added to the culture and theCHO cells express the nucleic acid which hybridized to the nucleic acidhaving the defined sequence or its complement. This invention furtherprovides a nucleic acid encoding a mammalian NPFF receptor, wherein thenucleic acid (a) hybridizes to a nucleic acid having the definedsequence shown in FIG. 4 (Seq. I.D. No. 3) under low stringencyconditions or a sequence complementary thereto and (b) is furthercharacterized by its ability to cause a change in the pH of a culture ofCHO cells when a NPFF peptide is added to the culture and the CHO cellsexpress the nucleic acid which hybridized to the nucleic acid having thedefined sequence or its complement. This invention also provides anucleic acid encoding a mammalian NPFF receptor, wherein the nucleicacid (a) hybridizes to a nucleic acid having the defined sequence shownin FIG. 7 (Seq. I.D. No. 5) under low stringency conditions or asequence complementary thereto and (b) is further characterized by itsability to cause a change in the pH of a culture of CHO cells when aNPFF peptide is added to the culture and the CHO cells express thenucleic acid which hybridized to the nucleic acid having the definedsequence or its complement.

[0124] This invention further provides a nucleic acid encoding amammalian NPFF receptor, wherein the nucleic acid (a) hybridizes to anucleic acid having the defined sequence shown in FIG. 11 (Seq. I.D. No.7) under low stringency conditions or a sequence complementary theretoand (b) is further characterized by its ability to cause a change in thepH of a culture of CHO cells when a NPFF peptide is added to the cultureand the CHO cells express the nucleic acid which hybridized to thenucleic acid having the defined sequence or its complement.

[0125] In one embodiment, the mammalian NPFF receptor is a rat NPFF1receptor. In another embodiment, the mammalian NPFF receptor is a humanNPFF1 receptor. In a further embodiment, the mammalian NPFF receptor isa human NPFF2 receptor. For purpose of the invention hybridization underlow stringency conditions means hybridization performed at 40° C. in ahybridization buffer containing 25% formamide, 5× SCC, 7 mM Tris, 1×Denhardt's, 25 μl/ml salmon sperm DNA. Wash at 40° C. in 0.1× SCC, 0.1%SDS. Changes in pH are measured through microphysiometric measurement ofreceptor mediated extracellular acidification rates. Because cellularmetabolism is intricately involved in a broad range of cellular events(including receptor activation of multiple messenger pathways), the useof microphysiometric measurements of cell metabolism can in principleprovide a generic assay of cellular activity arising from the activationof any receptor regardless of the specifics of the receptor's signalingpathway. General guidelines for transient receptor expression, cellpreparation and microphysiometric recording are described elsewhere(Salon, J. A. and Owicki, J. A., 1996). Receptors and/or control vectorsare transiently expressed in CHO-K1 cells, by liposome mediatedtransfection according to the manufacturers recommendations(LipofectAMINE, GibcoBRL, Gaithersburg, Md.), and maintained in Ham'sF-12 complete (10% serum). A total of 10 μg of DNA is used to transfecteach 75 cm² flask which had been split 24 hours prior to thetransfection and judged to be 70-80% confluent at the time oftransfection. 24 hours post transfection, the cells are harvested and3×10⁵ cells seeded into microphysiometer capsules. Cells are allowed toattach to the capsule membrane for an additional 24 hours; during thelast 16 hours, the cells are switched to serum-free F-12 complete tominimize ill-defined metabolic stimulation caused by assorted serumfactors. On the day of the experiment the cell capsules are transferredto the microphysiometer and allowed to equilibrate in recording media(low buffer RPMI 1640, no bicarbonate, no serum (Molecular DevicesCorporation, Sunnyvale, Calif.) containing 0.1% fatty acid free BSA),during which a baseline measurement of basal metabolic activity isestablished. A standard recording protocol specifies a 100 μl/min flowrate, with a 2 min total pump cycle which includes a 30 sec flowinterruption during which the acidification rate measurement is taken.Ligand challenges involve a 1 min 20 sec exposure to the sample justprior to the first post challenge rate measurement being taken, followedby two additional pump cycles for a total of 5 min 20 sec sampleexposure. Typically, drugs in a primary screen are presented to thecells at 10 μM final concentration. Ligand samples are then washed outand the acidification rates reported are expressed as a percentageincrease of the peak response over the baseline rate observed just priorto challenge. Endogenous NPFF peptides include rat NPFF (FLFQPQRF-NH2)and rat A18Fa (AGEGLSSPFWSLAAPQRF-NH2).

[0126] This invention provides a purified mammalian NPFF receptorprotein. In one embodiment, the purified mammalian NPFF receptor proteinis a human NPFF1 receptor protein. In another embodiment, the purifiedmammalian NPFF receptor protein is a rat NPFF1 receptor protein. In afurther embodiment, the purified mammalian NPFF receptor protein is ahuman NPFF2 receptor protein.

[0127] This invention provides a vector comprising nucleic acid encodinga mammalian NPFF receptor. In one embodiment, the mammalian NPFFreceptor protein is a NPFF1 receptor protein. In another embodiment ofthe present invention the mammalian NPFF receptor protein is a NPFF2receptor protein. In one embodiment, the mammalian NPFF receptor is arat NPFF1 receptor. In another embodiment, the mammalian NPFF receptoris a human NPFF1 receptor. In a further embodiment, the mammalian NPFFreceptor is a human NPFF2 receptor.

[0128] In an embodiment, the vector is adapted for expression in a cellwhich comprises the regulatory elements necessary for expression of thenucleic acid in the cell operatively linked to the nucleic acid encodingthe mammalian NPFF receptor as to permit expression thereof. In separateembodiments, the cell is a bacterial cell, an amphibian cell, a yeastcell, an insect cell or a mammalian cell. In another embodiment, thevector is a baculovirus. In one embodiment, the vector is a plasmid.

[0129] This invention provides a plasmid designated pEXJ-rNPFF1 (ATCCAccession No. 203184). This plasmid comprises the regulatory elementsnecessary for expression of DNA in a mammalian cell operatively linkedto DNA encoding the mammalian NPFF1 receptor so as to permit expressionthereof. This invention also provides a plasmid designated pWE15-hNPFF1(ATCC Accession No. 203183). This invention further provides a plasmiddesignated pCDNA3.1-hNPFF2b (ATCC Accession No. 203255). This inventionadditionally provides a plasmid designated pcDNA3.1-hNPFF1 (ATCCAccession No. 203605).

[0130] These plasmids (pEXJ-rNPFF1 and pWE15-hNPFF1) were deposited onSep. 9, 1998, with the American Type Culture Collection (ATCC), 10801University Blvd., Manassas, Va. 20110-2209, U.S.A. under the provisionsof the Budapest Treaty for the International Recognition of the Depositof Microorganisms for the Purposes of Patent Procedure and were accordedATCC Accession Nos. 203184 and 203183, respectively. PlasmidpCDNA3.1-hNPFF2b was deposited on Sep. 22, 1998, with the American TypeCulture Collection (ATCC), 10801 University Blvd., Manassas, Va.20110-2209, U.S.A. under the provisions of the Budapest Treaty for theInternational Recognition of the Deposit of Microorganisms for thePurposes of Patent Procedure and was accorded ATCC Accession No. 203255.Plasmid pcDNA3.1-hNPFF1 was deposited on Jan. 21, 1999, with theAmerican Type Culture Collection (ATCC), 10801 University Blvd.,Manassas, Va. 20110-2209, U.S.A. under the provisions of the BudapestTreaty for the International Recognition of the Deposit ofMicroorganisms for the Purposes of Patent Procedure and was accordedATCC Accession No. 203605.

[0131] This invention further provides for any vector or plasmid whichcomprises modified untranslated sequences, which are beneficial forexpression in desired host cells or for use in binding or functionalassays. For example, a vector or plasmid with untranslated sequences ofvarying lengths may express differing amounts of the polypeptidedepending upon the host cell used. In an embodiment, the vector orplasmid comprises the coding sequence of the polypeptide and theregulatory elements necessary for expression in the host cell.

[0132] This invention provides a cell comprising a vector comprising anucleic acid encoding the mammalian NPFF receptor. In an embodiment, thecell is a non-mammalian cell. In a further embodiment, the non-mammaliancell is a Xenopus oocyte cell or a Xenopus melanophore cell. In anotherembodment, the cell is a mammalian cell. In a further embodiment, themammalian cell is a COS-7 cell, a 293 human embryonic kidney cell, aNIH-3T3 cell, a LM(tk-) cell, a mouse Y1 cell, or a CHO cell.

[0133] This invention provides an insect cell comprising a vectoradapted for expression in an insect cell which comprises a nucleic acidencoding a mammalian NPFF receptor. In another embodiment, the insectcell is an Sf9 cell, an Sf21 cell or a Trichoplusia ni 5B1-4 (HighFive)cell.

[0134] This invention provides a membrane preparation isolated from anyone of the cells described above.

[0135] This invention provides a nucleic acid probe comprising at least15 nucleotides, which probe specifically hybridizes with a nucleic acidencoding a mammalian NPFF receptor, wherein the probe has a uniquesequence corresponding to a sequence present within one of the twostrands of the nucleic acid encoding the mammalian NPFF receptor and arecontained in plasmid pEXJ-rNPFF1, plasmid pWE15-hNPFF1, pCDNA3.1-hNPFF2bor pcDNA3.1-hNPFF1. This invention also provides a nucleic acid probecomprising at least 15 nucleotides, which probe specifically hybridizeswith a nucleic acid encoding a mammalian NPFF receptor, wherein theprobe has a unique sequence corresponding to a sequence present within(a) the nucleic acid sequence shown in FIG. 1 (Seq. I.D. No. 1) or (b)the reverse complement thereto. This invention also provides a nucleicacid probe comprising at least 15 nucleotides, which probe specificallyhybridizes with a nucleic acid encoding a mammalian NPFF receptor,wherein the probe has a unique sequence corresponding to a sequencepresent within (a) the nucleic acid sequence shown in FIG. 4 (Seq. I.D.No. 3) or (b) the reverse complement thereto. This invention alsoprovides a nucleic acid probe comprising at least 15 nucleotides, whichprobe specifically hybridizes with a nucleic acid encoding a mammalianNPFF receptor, wherein the probe has a unique sequence corresponding toa sequence present within (a) the nucleic acid sequence shown in FIG. 7(Seq. I.D. No. 5) or (b) the reverse complement thereto. This inventionalso provides a nucleic acid probe comprising at least 15 nucleotides,which probe specifically hybridizes with a nucleic acid encoding amammalian NPFF receptor, wherein the probe has a unique sequencecorresponding to a sequence present within (a) the nucleic acid sequenceshown in FIG. 11 (Seq. I.D. No. 7) or (b) the reverse complementthereto. In one embodiment, the nucleic acid is DNA. In anotherembodiment, the nucleic acid is RNA.

[0136] As used herein, the phrase “specifically hybridizing” means theability of a nucleic acid molecule to recognize a nucleic acid sequencecomplementary to its own and to form double-helical segments throughhydrogen bonding between complementary base pairs.

[0137] Nucleic acid probe technology is well known to those skilled inthe art who will readily appreciate that such probes may vary greatly inlength and may be labeled with a detectable label, such as aradioisotope or flourescent dye, to facilitate detection of the probe.DNA probe molecules may be produced by insertion of a DNA molecule whichencodes the polypeptides of this invention into suitable vectors, suchas plasmids or bacteriophages, followed by transforming into suitablebacterial host cells, replication in the transformed bacterial hostcells and harvesting of the DNA probes, using methods well known in theart. Alternatively, probes may be generated chemically from DNAsynthesizers.

[0138] RNA probes may be generated by inserting the DNA molecule whichencodes the polypeptides of this invention downstream of a bacteriophagepromoter such as T3, T7, or SP6. Large amounts of RNA probe may beproduced by incubating the labeled nucleotides with the linearizedfragment where it contains an upstream promoter in the presence of theappropriate RNA polymerase.

[0139] This invention provides an antisense oligonucleotide having asequence capable of specifically hybridizing to RNA encoding a mammalianNPFF receptor, so as to prevent translation of the RNA. This inventionalso provides an antisense oligonucleotide having a sequence capable ofspecifically hybridizing to genomic DNA encoding a mammalian NPFFreceptor, so as to prevent translation of the genomic DNA. In oneembodiment, the oligonucleotide comprises chemically modifiednucleotides or nucleotide analogues.

[0140] This invention provides an antibody capable of binding to amammalian NPFF receptor encoded by a nucleic acid encoding a mammalianNPFF receptor. In one embodiment, the mammalian NPFF receptor is a ratNPFF1 receptor. In another embodiment, the mammalian NPFF receptor is ahuman NPFF1 receptor. In a further embodiment, the mammalian NPFFreceptor is a human NPFF2 receptor. This invention also provides anagent capable of competitively inhibiting the binding of the antibody toa mammalian NPFF receptor In one embodiment, the antibody is amonoclonal antibody or antisera.

[0141] This invention provides a pharmaceutical composition comprising(a) an amount of the oligonucleotide capable of passing through a cellmembrane and effective to reduce expression of a mammalian NPFF receptorand (b) a pharmaceutically acceptable carrier capable of passing throughthe cell membrane. In an embodiment, the oligonucleotide is coupled to asubstance which inactivates mRNA. In a further embodiment, the substancewhich inactivates mRNA is a ribozyme. In another embodiment, thepharmaceutically acceptable carrier comprises a structure which binds toa mammalian NPFF receptor on a cell capable of being taken up by thecells after binding to the structure. In a further embodiment, thepharmaceutically acceptable carrier is capable of binding to a mammalianNPFF receptor which is specific for a selected cell type.

[0142] This invention provides a pharmaceutical composition whichcomprises an amount of an antibody effective to block binding of aligand to a human NPFF receptor and a pharmaceutically acceptablecarrier.

[0143] As used herein, the phrase “pharmaceutically acceptable carrier”means any of the standard pharmceutically acceptable carriers. Examplesinclude, but are not limited to, phosphate buffered saline,physiological saline, water, and emulsions, such as oil/water emulsions.

[0144] This invention provides a transgenic, nonhuman mammal expressingDNA encoding a mammalian NPFF receptor. This invention also provides atransgenic, nonhuman mammal comprising a homologous recombinationknockout of the native mammalian NPFF receptor. This invention furtherprovides a transgenic, nonhuman mammal whose genome comprises antisenseDNA complementary to the DNA encoding a mammalian NPFF receptor soplaced within the genome as to be transcribed into antisense mRNA whichis complementary to mRNA encoding the mammalian NPFF receptor and whichhybridizes to mRNA encoding the mammalian NPFF receptor, therebyreducing its translation. In an embodiment, the DNA encoding themammalian NPFF receptor additionally comprises an inducible promoter. Inanother embodiment, the DNA encoding the mammalian NPFF receptoradditionally comprises tissue specific regulatory elements. In a furtherembodiment, the transgenic, nonhuman mammal is a mouse.

[0145] Animal model systems which elucidate the physiological andbehavioral roles of the polypeptides of this invention are produced bycreating transgenic animals in which the activity of the polypeptide iseither increased or decreased, or the amino acid sequence of theexpressed polypeptide is altered, by a variety of techniques. Examplesof these techniques include, but are not limited to: 1) Insertion ofnormal or mutant versions of DNA encoding the polypeptide, bymicroinjection, electroporation, retroviral transfection or other meanswell known to those in the art, into appropriate fertilized embryos inorder to produce a transgenic animal or 2) Homologous recombination ofmutant or normal, human or animal versions of these genes with thenative gene locus in transgenic animals to alter the regulation ofexpression or the structure of these polypeptide sequences. Thetechnique of homologous recombination is well known in the art. Itreplaces the native gene with the inserted gene and so is useful forproducing an animal that cannot express native polypeptides but doesexpress, for example, an inserted mutant polypeptide, which has replacedthe native polypeptide in the animal's genome by recombination,resulting in underexpression of the transporter. Microinjection addsgenes to the genome, but does not remove them, and so is useful forproducing an animal which expresses its own and added polypeptides,resulting in overexpression of the polypeptides.

[0146] One means available for producing a transgenic animal, with amouse as an example, is as follows: Female mice are mated, and theresulting fertilized eggs are dissected out of their oviducts. The eggsare stored in an appropriate medium such as M2 medium. DNA or cDNAencoding a polypeptide of this invention is purified from a vector bymethods well known in the art. Inducible promoters may be fused with thecoding region of the DNA to provide an experimental means to regulateexpression of the trans-gene. Alternatively, or in addition, tissuespecific regulatory elements may be fused with the coding region topermit tissue-specific expression of the trans-gene. The DNA, in anappropriately buffered solution, is put into a microinjection needle(which may be made from capillary tubing using a pipet puller) and theegg to be injected is put in a depression slide. The needle is insertedinto the pronucleus of the egg, and the DNA solution is injected. Theinjected egg is then transferred into the oviduct of a pseudopregnantmouse (a mouse stimulated by the appropriate hormones to maintainpregnancy but which is not actually pregnant ), where it proceeds to theuterus, implants, and develops to term. As noted above, microinjectionis not the only method for inserting DNA into the egg cell, and is usedhere only for exemplary purposes.

[0147] This invention provides a process for identifying a chemicalcompound which specifically binds to a mammalian NPFF receptor whichcomprises contacting cells containing DNA encoding and expressing ontheir cell surface the mammalian NPFF receptor, wherein such cells donot normally express the mammalian NPFF receptor, with the compoundunder conditions suitable for binding, and detecting specific binding ofthe chemical compound to the mammalian NPFF receptor. This inventionalso provides a process for identifying a chemical compound whichspecifically binds to a mammalian NPFF receptor which comprisescontacting a membrane fraction from a cell extract of cells containingDNA encoding and expressing on their cell surface the mammalian NPFFreceptor, wherein such cells do not normally express the mammalian NPFFreceptor, with the compound under conditions suitable for binding, anddetecting specific binding of the chemical compound to the mammalianNPFF receptor. In one embodiment, the NPFF receptor is a NPFF1 receptor.In a further embodiment, the mammalian NPFF1 receptor is a rat NPFF1receptor. In another embodiment, the mammalian NPFF1 receptor is a humanNPFF1 receptor. In one embodiment, the mammalian NPFF receptor is aNPFF2 receptor. In a further embodiment, the mammalian NPFF2 receptor isa human NPFF2 receptor. In another embodiment, the mammalian NPFFreceptor has substantially the same amino acid sequence as the NPFFreceptor encoded by plasmid pEXJ-rNPFF1, plasmid pWE15-hNPFF1, plasmidpCDNA3.1-hNPFF2b or plasmid pcDNA3.1-hNPFF1. In a further embodiment,the mammalian NPFF receptor has substantially the same amino acidsequence as that shown in FIG. 2 (Seq. I.D. No. 2), FIG. 5 (Seq. I.D.No. 4), FIG. 8 (Seq. I.D. No. 6), or FIG. 12 (Seq. I.D. No. 8). Inanother embodiment, the mammalian NPFF receptor has the amino acidsequence shown in FIG. 2 (Seq. I.D. No. 2), FIG. 5 (Seq. I.D. No. 4)FIG. 8 (Seq. I.D. No. 6), or FIG. 12 (Seq. I.D. No. 8). In oneembodiment, the compound is not previously known to bind to a mammalianNPFF receptor. This invention further provides a compound identified bythe above-described processes.

[0148] In one embodiment of the above-described processes, the cell isan insect cell. In another embodiment, the cell is a mammalian cell. Ina further embodiment, the cell is nonneuronal in origin. In a furtherembodiment, the nonneuronal cell is a COS-7 cell, 293 human embryonickidney cell, a CHO cell, a NIH-3T3 cell, a mouse Y1 cell, or a LM(tk-)cell. In an embodiment, the compound is a compound not previously knownto bind to a mammalian NPFF receptor. This invention also provides acompound identified by the above-described process.

[0149] This invention provides a process involving competitive bindingfor identifying a chemical compound which specifically binds to amammalian NPFF receptor which comprises separately contacting cellsexpressing on their cell surface the mammalian NPFF receptor, whereinsuch cells do not normally express the mammalian NPFF receptor, withboth the chemical compound and a second chemical compound known to bindto the receptor, and with only the second chemical compound, underconditions suitable for binding of both compounds, and detectingspecific binding of the chemical compound to the mammalian NPFFreceptor, a decrease in the binding of the second chemical compound tothe mammalian NPFF receptor in the presence of the chemical compoundindicating that the chemical compound binds to the mammalian NPFFreceptor.

[0150] This invention also provides a process involving competitivebinding for identifying a chemical compound which specifically binds toa mammalian NPFF receptor which comprises separately contacting amembrane preparation from cells expressing on their cell surface themammalian NPFF receptor, wherein such cells do not normally express themammalian NPFF receptor, with both the chemical compound and a secondchemical compound known to bind to the receptor, and with only thesecond chemical compound, under conditions suitable for binding of bothcompounds, and detecting specific binding of the chemical compound tothe mammalian NPFF receptor, a decrease in the binding of the secondchemical compound to the mammalian NPFF receptor in the presence of thechemical compound indicating that the chemical compound binds to themammalian NPFF receptor.

[0151] In one embodiment, the mammalian NPFF receptor is a NPFF1receptor. In a further embodiment, the mammalian NPFF1 receptor is a ratNPFF1 receptor. In another embodiment, the mammalian NPFF1 receptor is ahuman NPFF1 receptor. In another embodiment, the mammalian NPFF receptoris a NPFF2 receptor. In a further embodiment, the NPFF2 receptor is ahuman NPFF2 receptor. In another embodiment, the mammalian NPFF receptorhas substantially the same amino acid sequence as the NPFF receptorencoded by plasmid pEXJ-rNPFF1, pWE15-hNPFF1, pCDNA3.1-hNPFF2b orpcDNA3.1-hNPFF1. In a further embodiment, the mammalian NPFF receptorhas substantially the same amino acid sequence as that shown in FIG. 2(Seq. I.D. No. 2), FIG. 5 (Seq. I.D. No. 4), FIG. 8 (Seq. I.D. No. 6) orFIG. 12 (Seq. I.D. No. 8). In another embodiment, the mammalian NPFFreceptor has the amino acid sequence shown in FIG. 2 (Seq. I.D. No. 2),FIG. 5 (Seq. I.D. No. 4), FIG. 8 (Seq. I.D. No. 6) or FIG. 12 (Seq. I.D.No. 8).

[0152] In one embodiment, the cell is an insect cell. In anotherembodiment, the cell is a mammalian cell. In a further embodiment, thecell is nonneuronal in origin. In another embodiment, the nonneuronalcell is a COS-7 cell, 293 human embryonic kidney cell, a CHO cell, aNIH-3T3 cell, a mouse Y1 cell, or a LM(tk-) cell. In one embodiment, thecompound is not previously known to bind to a mammalian NPFF receptor.

[0153] This invention provides a compound identified by theabove-described processes.

[0154] This invention provides a method of screening a plurality ofchemical compounds not known to bind to a mammalian NPFF receptor toidentify a compound which specifically binds to the mammalian NPFFreceptor, which comprises (a) contacting cells transfected with andexpressing DNA encoding the mammalian NPFF receptor with a compoundknown to bind specifically to the mammalian NPFF receptor; (b)contacting the preparation of step (a) with the plurality of compoundsnot known to bind specifically to the mammalian NPFF receptor, underconditions permitting binding of compounds known to bind the mammalianNPFF receptor; (c) determining whether the binding of the compound knownto bind to the mammalian NPFF receptor is reduced in the presence of thecompounds within the plurality of compounds, relative to the binding ofthe compound in the absence of the plurality of compounds; and if so (d)separately determining the binding to the mammalian NPFF receptor ofcompounds included in the plurality of compounds, so as to therebyidentify the compound which specifically binds to the mammalian NPFFreceptor.

[0155] This invention provides a method of screening a plurality ofchemical compounds not known to bind to a mammalian NPFF receptor toidentify a compound which specifically binds to the mammalian NPFFreceptor, which comprises (a) contacting a membrane preparation fromcells transfected with and expressing DNA encoding the mammalian NPFFreceptor with the plurality of compounds not known to bind specificallyto the mammalian NPFF receptor under conditions permitting binding ofcompounds known to bind to the mammalian NPFF receptor; (b) determiningwhether the binding of a compound known to bind to the mammalian NPFFreceptor is reduced in the presence of any compound within the pluralityof compounds, relative to the binding of the compound in the absence ofthe plurality of compounds; and if so (c) separately determining thebinding to the mammalian NPFF receptor of compounds included in theplurality of compounds, so as to thereby identify the compound whichspecifically binds to the mammalian NPFF receptor.

[0156] This invention provides a method of screening a plurality ofchemical compounds not known to bind to a mammalian NPFF receptor toidentify a compound which specifically binds to the mammalian NPFFreceptor, which comprises (a) contacting a membrane preparation fromcells transfected with and expressing the mammalian NPFF receptor with acompound known to bind specifically to the mammalian NPFF receptor; (b)contacting the preparation of step (a) with the plurality of compoundsnot known to bind specifically to the mammalian NPFF receptor, underconditions permitting binding of compounds known to bind the mammalianNPFF receptor; (c) determining whether the binding of the compound knownto bind to the mammalian NPFF receptor is reduced in the presence of thecompounds within the plurality of compounds, relative to the binding ofthe compound in the absence of the plurality of compounds; and if so (d)separately determining the binding to the mammalian NPFF receptor ofcompounds included in the plurality of compounds, so as to therebyidentify the compound which specifically binds to the mammalian NPFFreceptor.

[0157] In one embodiment of the above-described methods, the mammalianNPFF receptor is a NPFF1 receptor. In a further embodiment, themammalian NPFF1 receptor is a rat NPFF1 receptor. In another embodiment,the mammalian NPFF1 receptor is a human NPFF1 receptor. In anotherembodiment, the mammalian NPFF receptor is a NPFF2 receptor. In afurther embodiment the NPFF2 receptor is a human NPFF2 receptor. Inanother embodiment, the cell is a mammalian cell. In a furtherembodiment, the mammalian cell is non-neuronal in origin. In anotherembodiment, the non-neuronal cell is a COS-7 cell. a 293 human embryonickidney cell, a LM(tk-) cell, a CHO cell, a mouse Y1 cell, or an NIH-3T3cell.

[0158] This invention also provides a method of detecting expression ofa mammalian NPFF receptor by detecting the presence of mRNA coding forthe mammalian NPFF receptor which comprises obtaining total mRNA fromthe cell and contacting the mRNA so obtained from a nucleic acid probeunder hybridizing conditions, detecting the presence of mRNA hybridizingto the probe, and thereby detecting the expression of the mammalian NPFFreceptor by the cell.

[0159] This invention further provides a method of detecting thepresence of a mammalian NPFF receptor on the surface of a cell whichcomprises contacting the cell with an antibody under conditionspermitting binding of the antibody to the receptor, detecting thepresence of the antibody bound to the cell, and thereby detecting thepresence of the mammalian NPFF receptor on the surface of the cell.

[0160] This invention provides a method of determining the physiologicaleffects of varying levels of activity of mammalian NPFF receptors whichcomprises producing a transgenic, nonhuman mammal whose levels ofmammalian NPFF receptor activity are varied by use of an induciblepromoter which regulates mammalian NPFF receptor expression.

[0161] This invention also provides a method of determining thephysiological effects of varying levels of activity of mammalian NPFFreceptors which comprises producing a panel of transgenic, nonhumanmammals each expressing a different amount of mammalian NPFF receptor.

[0162] This invention provides a method for identifying an antagonistcapable of alleviating an abnormality wherein the abnormality isalleviated by decreasing the activity of a mammalian NPFF receptorcomprising administering a compound to a transgenic, nonhuman mammal,and determining whether the compound alleviates the physical andbehavioral abnormalities displayed by the transgenic, nonhuman mammal asa result of overactivity of a mammalian NPFF receptor, the alleviationof the abnormality identifying the compound as an antagonist. Thisinvention also provides an antagonist identified by the above-describedmethod. This invention further provides a pharmaceutical compositioncomprising an antagonist identified by the above-described method and apharmaceutically acceptable carrier. This invention provides a method oftreating an abnormality in a subject wherein the abnormality isalleviated by decreasing the activity of a mammalian NPFF receptor whichcomprises administering to the subject an effective amount of thispharmaceutical composition, thereby treating the abnormality.

[0163] This invention provides a method for identifying an agonistcapable of alleviating an abnormality in a subject wherein theabnormality is alleviated by increasing the activity of a mammalian NPFFreceptor comprising administering a compound to transgenic, nonhumanmammal, and determining whether the compound alleviates the physical andbehavioral abnormalities displayed by the transgenic, nonhuman mammal,the alleviation of the abnormality identifying the compound as anagonist. This invention also provides an agonist identified by theabove-described method. This invention further provides a pharmaceuticalcomposition comprising an agonist identified by the above-describedmethod and a pharmaceutically acceptable carrier. This invention furtherprovides a method of treating an abnormality in a subject wherein theabnormality is alleviated by increasing the activity of a mammalian NPFFreceptor which comprises administering to the subject an effectiveamount of this pharmaceutical composition, thereby treating theabnormality.

[0164] This invention provides a method for diagnosing a predispositionto a disorder associated with the activity of a specific mammalianallele which comprises: (a) obtaining DNA of subjects suffering from thedisorder; (b) performing a restriction digest of the DNA with a panel ofrestriction enzymes; (c) electrophoretically separating the resultingDNA fragments on a sizing gel; (d) contacting the resulting gel with anucleic acid probe capable of specifically hybridizing with a uniquesequence included within the sequence of a nucleic acid moleculeencoding a mammalian NPFF receptor and labeled with a detectable marker;(e) detecting labeled bands which have hybridized to the DNA encoding amammalian NPFF receptor labeled with a detectable marker to create aunique band pattern specific to the DNA of subjects suffering from thedisorder; (f) preparing DNA obtained for diagnosis by steps (a)-(e); and(g) comparing the unique band pattern specific to the DNA of subjectssuffering from the disorder from step (e) and the DNA obtained fordiagnosis from step (f) to determine whether the patterns are the sameor different and to diagnose thereby predisposition to the disorder ifthe patterns are the same. In one embodiment, a disorder associated withthe activity of a specific mammalian allele is diagnosed.

[0165] This invention provides a method of preparing the purifiedmammalian NPFF receptor which comprises: (a) inducing cells to expressthe mammalian NPFF receptor; (b) recovering the mammalian NPFF receptorfrom the induced cells; and (c) purifying the mammalian NPFF receptor sorecovered.

[0166] This invention provides a method of preparing the purifiedmammalian NPFF receptor which comprises: (a) inserting nucleic acidencoding the mammalian NPFF receptor in a suitable vector; (b)introducing the resulting vector in a suitable host cell; (c) placingthe resulting cell in suitable condition permitting the production ofthe isolated mammalian NPFF receptor; (d) recovering the mammalian NPFFreceptor produced by the resulting cell; and (e) purifying the mammalianNPFF receptor so recovered.

[0167] This invention provides a process for determining whether achemical compound is a mammalian NPFF receptor agonist which comprisescontacting cells transfected with and expressing DNA encoding themammalian NPFF receptor with the compound under conditions permittingthe activation of the mammalian NPFF receptor, and detecting an increasein mammalian NPFF receptor activity, so as to thereby determine whetherthe compound is a mammalian NPFF receptor agonist. This invention alsoprovides a process for determining whether a chemical compound is amammalian NPFF1 receptor antagonist which comprises contacting cellstransfected with and expressing DNA encoding the mammalian NPFF receptorwith the compound in the presence of a known mammalian NPFF receptoragonist, under conditions permitting the activation of the mammalianNPFF receptor, and detecting a decrease in mammalian NPFF receptoractivity, so as to thereby determine whether the compound is a mammalianNPFF receptor antagonist. In one embodiment, the mammalian NPFF receptoris a NPFF1 receptor. In a further embodiment, the mammalian NPFF1receptor is a rat NPFF1 receptor. In another embodiment, the mammalianNPFF1 receptor is a human NPFF1 receptor. In one embodiment, themammalian NPFF receptor is a NPFF2 receptor. In a further embodiment,the mammalian NPFF2 receptor is a human NPFF2 receptor.

[0168] This invention further provides a pharmaceutical compositionwhich comprises an amount of a mammalian NPFF receptor agonistdetermined by the above-described process effective to increase activityof a mammalian NPFF receptor and a pharmaceutically acceptable carrier.In one embodiment, the mammalian NPFF receptor agonist is not previouslyknown.

[0169] This invention provides a pharmaceutical composition whichcomprises an amount of a mammalian NPFF receptor antagonist determinedby the above-described process effective to reduce activity of amammalian NPFF receptor and a pharmaceutically acceptable carrier. Inone embodiment, the mammalian NPFF receptor antagonist is not previouslyknown.

[0170] This invention provides a process for determining whether achemical compound specifically binds to and activates a mammalian NPFFreceptor, which comprises contacting cells producing a second messengerresponse and expressing on their cell surface the mammalian NPFFreceptor, wherein such cells do not normally express the mammalian NPFFreceptor, with the chemical compound under conditions suitable foractivation of the mammalian NPFF receptor, and measuring the secondmessenger response in the presence and in the absence of the chemicalcompound, a change in the second messenger response in the presence ofthe chemical compound indicating that the compound activates themammalian NPFF receptor. In one embodiment, the second messengerresponse comprises chloride channel activation and the change in secondmessenger is an increase in the level of inward chloride current.

[0171] This invention also provides a process for determining whether achemical compound specifically binds to and inhibits activation of amammalian NPFF receptor, which comprises separately contacting cellsproducing a second messenger response and expressing on their cellsurface the mammalian NPFF receptor, wherein such cells do not normallyexpress the mammalian NPFF receptor, with both the chemical compound anda second chemical compound known to activate the mammalian NPFFreceptor, and with only the second chemical compound, under conditionssuitable for activation of the mammalian NPFF receptor, and measuringthe second messenger response in the presence of only the secondchemical compound and in the presence of both the second chemicalcompound and the chemical compound, a smaller change in the secondmessenger response in the presence of both the chemical compound and thesecond chemical compound than in the presence of only the secondchemical compound indicating that the chemical compound inhibitsactivation of the mammalian NPFF receptor. In one embodiment, the secondmessenger response comprises chloride channel activation and the changein second messenger response is a smaller increase in the level ofinward chloride current in the presence of both the chemical compoundand the second chemical compound than in the presence of only the secondchemical compound. This invention also provides the above-describedprocesses performed with membrane preparations from cells producing asecond messenger response and transfected with and expressing themammalian NPFF receptor.

[0172] In one embodiment of the above-described processes, the mammalianNPFF receptor is a NPFF1 receptor. In a further embodiment, themammalian NPFF1 receptor is a rat NPFF1 receptor. In another embodiment,the mammalian NPFF1 receptor is a human NPFF1 receptor. In anotherembodiment, the mammalian NPFF receptor is a NPFF2 receptor. In afurther embodiment, the mammalian NPFF2 receptor is a human NPFF2receptor. In another embodiment, the mammalian NPFF receptor hassubstantially the same amino acid sequence as encoded by the plasmidpEXJ-rNPFF1, pWE15-hNPFF1, pCDNA3.1-hNPFF2b or pcDNA3.1-hNPFF1. In afurther embodiment, the mammalian NPFF receptor has substantially thesame amino acid sequence as that shown in FIG. 2 (Seq. I.D. No. 2), FIG.5 (Seq. I.D. No. 4), FIG. 8 (Seq. I.D. No. 6) or FIG. 12 (Seq. I.D. No.8). In another embodiment, the mammalian NPFF receptor has an amino acidsequence identical to the amino acid sequence shown in FIG. 2 (Seq. I.D.No. 2), FIG. 5 (Seq. I.D. No. 4), FIG. 8 (Seq. I.D. No. 6) or FIG. 12(Seq. I.D. No. 8). In an embodiment, the cell is an insect cell. In afurther embodiment, the cell is a mammalian cell. In a still furtherembodiment, the mammalian cell is nonneuronal in origin. In anotherembodiment, the nonneuronal cell is a COS-7 cell CHO cell, 293 humanembryonic kidney cell, NIH-3T3 cell or LM(tk-) cell. In an embodiment,the compound is not previously known to bind to a mammalian NPFFreceptor. This invention also provides a compound determined by theabove-described processes.

[0173] This invention also provides a pharmaceutical composition whichcomprises an amount of a mammalian NPFF receptor agonist determined bythe above-described processes effective to increase activity of amammalian NPFF receptor and a pharmaceutically acceptable carrier. Inone embodiment, the mammalian NPFF receptor agonist is not previouslyknown.

[0174] This invention further provides a pharmaceutical compositionwhich comprises an amount of a mammalian NPFF receptor antagonistdetermined by the above-described processes effective to reduce activityof a mammalian NPFF receptor and a pharmaceutically acceptable carrier.In one embodiment, the mammalian NPFF receptor antagonist is notpreviously known.

[0175] This invention provides a method of screening a plurality ofchemical compounds not known to activate a mammalian NPFF receptor toidentify a compound which activates the mammalian NPFF receptor whichcomprises: (a) contacting cells transfected with and expressing themammalian NPFF receptor with the plurality of compounds not known toactivate the mammalian NPFF receptor, under conditions permittingactivation of the mammalian NPFF receptor; (b) determining whether theactivity of the mammalian NPFF receptor is increased in the presence ofthe compounds; and if so (c) separately determining whether theactivation of the mammalian NPFF receptor is increased by each compoundincluded in the plurality of compounds, so as to thereby identify thecompound which activates the mammalian NPFF receptor. In one embodiment,the mammalian NPFF receptor is a human NPFF receptor. In a furtherembodiment the human NPFF receptor is a human NPFF1 receptor or a humanNPFF2 receptor.

[0176] This invention provides a method of screening a plurality ofchemical compounds not known to inhibit the activation of a mammalianNPFF receptor to identify a compound which inhibits the activation ofthe mammalian NPFF receptor, which comprises: (a) contacting cellstransfected with and expressing the mammalian NPFF receptor with theplurality of compounds in the presence of a known mammalian NPFFreceptor agonist, under conditions permitting activation of themammalian NPFF receptor; (b) determining whether the activation of themammalian NPFF receptor is reduced in the presence of the plurality ofcompounds, relative to the activation of the mammalian NPFF receptor inthe absence of the plurality of compounds; and if so (c) separatelydetermining the inhibition of activation of the mammalian NPFF receptorfor each compound included in the plurality of compounds, so as tothereby identify the compound which inhibits the activation of themammalian NPFF receptor. In one embodiment, the mammalian NPFF receptoris a NPFF1 receptor. In a further embodiment, the mammalian NPFF1receptor is a rat NPFF1 receptor. In another embodiment, the NPFF1receptor is a human NPFF1 receptor. In another embodiment, the mammalianNPFF receptor is a NPFF2 receptor. In a further embodiment, the NPFF2receptor is a human NPFF2 receptor.

[0177] In one embodiment of the above-described methods, the cell is amammalian cell. In another embodiment, the mammalian cell isnon-neuronal in origin. In a further embodiment, the non-neuronal cellis a COS-7 cell, a 293 human embryonic kidney cell, a LM(tk-) cell or anNIH-3T3 cell.

[0178] This invention provides a pharmaceutical composition comprising acompound identified by the above-described methods effective to increasemammalian NPFF receptor activity and a pharmaceutically acceptablecarrier.

[0179] This invention also provides a pharmaceutical compositioncomprising a compound identified by the above-described methodseffective to decrease mammalian NPFF receptor activity and apharmaceutically acceptable carrier.

[0180] This invention further provides a method of measuring polypeptideactivation in an oocyte expression system such as a Xenopus oocyteexpression system or melanophore. In an embodiment, polypeptideactivation is determined by measurement of ion channel activity. Inanother embodiment, polypeptide activation is measured by aequerinluminescence.

[0181] Expression of genes in Xenopus oocytes is well known in -he art(Coleman, A., 1984; Masu, Y., et al., 1994) and is performed usingmicroinjection of native mRNA or in vitro synthesized mRNA into frogoocytes. The preparation of in vitro synthesized mRNA can be performedby various standard techniques (Sambrook, et al. 1989) including usingT7 polymerase with the mCAP RNA mapping kit (Stratagene).

[0182] This invention provides a method of treating an abnormality in asubject wherein the abnormality is alleviated by increasing the activityof a mammalian NPFF receptor which comprises administering to thesubject an amount of a compound which is a mammalian NPFF receptoragonist effective to treat the abnormality. In separate embodiments, theabnormality is a regulation of a steroid hormone disorder, anepinephrine release disorder, a gastrointestinal disorder, acardiovascular disorder, an electrolyte balance disorder, hypertension,diabetes, a respiratory disorder, asthma, a reproductive functiondisorder, an immune disorder, an endocrine disorder, a musculoskeletaldisorder, a neuroendocrine disorder, a cognitive disorder, a memorydisorder, a sensory modulation and transmission disorder, a motorcoordination disorder, a sensory integration disorder, a motorintegration disorder, a dopaminergic function disorder, an appetitedisorder, obesity, a sensory transmission disorder, an olfactiondisorder, a sympathetic innervation disorder, an affective disorder,pain, psychotic behavior, morphine tolerance, opiate addiction ormigraine.

[0183] This invention provides a method of treating an abnormality in asubject wherein the abnormality is alleviated by decreasing the activityof a mammalian NPFF receptor which comprises administering to thesubject an amount of a compound which is a mammalian NPFF receptorantagonist effective to treat the abnormality. In separate embodiments,the abnormality is a regulation of a steroid hormone disorder, anepinephrine release disorder, a gastrointestinal disorder, acardiovascular disorder, an electrolyte balance disorder, hypertension,diabetes, a respiratory disorder, asthma, a reproductive functiondisorder, an immune disorder, an endocrine disorder, a musculoskeletaldisorder, a neuroendocrine disorder, a cognitive disorder, a memorydisorder, a sensory modulation and transmission disorder, a motorcoordination disorder, a sensory integration disorder, a motorintegration disorder, a dopaminergic function disorder, an appetitedisorder, obesity, a sensory transmission disorder, an olfactiondisorder, a sympathetic innervation disorder, an affective disorder,pain psychotic behavior, morphine tolerance, opiate addiction ormigraine.

[0184] This invention also provides the use of mammalian NPFF receptorsfor analgesia.

[0185] This invention provides a process for making a composition ofmatter which specifically binds to a mammalian NPFF receptor whichcomprises identifying a chemical compound using any of the processesdescribed herein for identifying a compound which binds to and/oractivates or inhibits activation of a mammalian NPFF receptor and thensynthesizing the chemical compound or a novel structural and functionalanalog or homolog thereof. In one embodiment, the mammalian NPFFreceptor is a human NPFF1 receptor. In another embodiment, the mammalianNPFF receptor is a human NPFF2 receptor.

[0186] This invention further provides a process for preparing apharmaceutical composition which comprises admixing a pharmaceuticallyacceptable carrier and a pharmaceutically acceptable amount of achemical compound identified by any of the processes described hereinfor identifying a compound which binds to and/or activates or inhibitsactivation of a mammalian NPFF receptor or a novel structural andfunctional analog or homolog thereof. In one embodiment, the mammalianNPFF receptor is a human NPFF1 receptor. In another embodiment, themammalian NPFF receptor is a human NPFF2 receptor.

[0187] Thus, once the gene for a targeted receptor subtype is cloned, itis placed into a recipient cell which then expresses the targetedreceptor subtype on its surface. This cell, which expresses a singlepopulation of the targeted human receptor subtype, is then propagatedresulting in the establishment of a cell line. This cell line, whichconstitutes a drug discovery system, is used in two different types ofassays: binding assays and functional assays. In binding assays, theaffinity of a compound for both the receptor subtype that is the targetof a particular drug discovery program and other receptor subtypes thatcould be associated with side effects are measured. These measurementsenable one to predict the potency of a compound, as well as the degreeof selectivity that the compound has for the targeted receptor subtypeover other receptor subtypes. The data obtained from binding assays alsoenable chemists to design compounds toward or away from one or more ofthe relevant subtypes, as appropriate, for optimal therapeutic efficacy.In functional assays, the nature of the response of the receptor subtypeto the compound is determined. Data from the functional assays showwhether the compound is acting to inhibit or enhance the activity of thereceptor subtype, thus enabling pharmacologists to evaluate compoundsrapidly at their ultimate human receptor subtypes targets permittingchemists to rationally design drugs that will be more effective and havefewer or substantially less severe side effects than existing drugs.

[0188] Approaches to designing and synthesizing receptorsubtype-selective compounds are well known and include traditionalmedicinal chemistry and the newer technology of combinatorial chemistry,both of which are supported by computer-assisted molecular modeling.With such approaches, chemists and pharmacologists use their knowledgeof the structures of the targeted receptor subtype and compoundsdetermined to bind and/or activate or inhibit activation of the receptorsubtype to design and synthesize structures that will have activity atthese receptor subtypes.

[0189] Combinatorial chemistry involves automated synthesis of a varietyof novel compounds by assembling them using different combinations ofchemical building blocks. The use of combinatorial chemistry greatlyaccelerates the process of generating compounds. The resulting arrays ofcompounds are called libraries and are used to screen for compounds(“lead compounds”) that demonstrate a sufficient level of activity atreceptors of interest. Using combinatorial chemistry it is possible tosynthesize “focused” libraries of compounds anticipated to be highlybiased toward the receptor target of interest.

[0190] Once lead compounds are identified, whether through the use ofcombinatorial chemistry or traditional medicinal chemistry or otherwise,a variety of homologs and analogs are prepared to facilitate anunderstanding of the relationship between chemical structure andbiological or functional activity. These studies define structureactivity relationships which are then used to design drugs with improvedpotency, selectivity and pharmacokinetic properties. Combinatorialchemistry is also used to rapidly generate a variety of structures forlead optimization. Traditional medicinal chemistry, which involves thesynthesis of compounds one at a time, is also used for furtherrefinement and to generate compounds not accessible by automatedtechniques. Once such drugs are defined the production is scaled upusing standard chemical manufacturing methodologies utilized throughoutthe pharmaceutical and chemistry industry.

[0191] This invention will be better understood from the ExperimentalDetails which follow. However, one skilled in the art will readilyappreciate that the specific methods and results discussed are merelyillustrative of the invention as described more fully in the claimswhich follow thereafter.

[0192] Experimental Details

[0193] Materials and Methods

[0194] Cloning of Rat and Human NPFF1 Receptor

[0195] MOPAC (Mixed Oligonucleotide Primed Amplification of cDNA

[0196] 100 ng of rat genomic DNA (Clonetech, Palo Alto, Calif.) was usedfor degenerate MOPAC PCR using Taq DNA polymerase (Boehringer-Mannheim,Indianapolis, Ind.) and the following degenerate oligonucleotides:JAB126, designed based on an alignment of the sixth transmembrane domainof more than 180 members of the rhodopsin superfamily of Gprotein-coupled receptors; and JAB108, designed based on an alignment ofthe seventh transmembrane domain of the same rhodopsin superfamily.

[0197] The conditions for the MOPAC PCR reaction were as follows: 3minute hold at 94° C.; 10 cycles of 1 minute at 94° C., 1 minute 45seconds at 44° C., 2 minutes at 72° C.; 30 cycles of 94° C. for 1minute, 49° C. for 1 minute 45 seconds, 2 minutes at 72° C.; 4 minutehold at 72° C.; 4° C. until ready for agarose gel electrophoresis.

[0198] The products were run on a 1% agarose TAE gel and bands of theexpected size (˜150 bp) were cut from the gel, purified using theQIAQUICK gel extraction kit (QIAGEN, Chatsworth, Calif.), and subclonedinto the TA cloning vector (Invitrogen, San Diego, Calif.). White(insert-containing) colonies were picked and subjected to PCR usingpCR2.1 vector primers JAB1 and JAB2 using the Expand Long Template PCRSystem and the following protocol: 94° C. hold for 3 minutes; 35 cyclesof 94° C. for 1 minute, 68° C. for 1 minute 15 seconds; 2 minute hold at68° C., 4° C. hold until products were ready for purification. PCRproducts were purified by isopropanol precipitation (10 μl PCR product,18 μl low TE, 10.5 μl 2M NaClO₄ and 21.5 μl isopropanol) and sequencedusing the ABI Big Dye cycle sequencing protocol and ABI 377 sequencers(ABI, Foster City, Calif.). Nucleotide and amino acid sequence analyseswere performed using the Wisconsin Package (GCG, Genetics ComputerGroup, Madison, Wis.). Two PCR products produced from rat genomic cDNA(MPR3-RGEN-31 and MPR3-RGEN-45) were determined to be identical clonesof a novel G protein-coupled receptor-like sequence based on databasesearches and its homology to other known G protein-coupled receptors(˜30-40% amino acid identity to dopamine D2, orexin, galanin,angiotensin 1 and 5-HT_(2b) receptors). This novel sequence wasdesignated SNORF2.

[0199] Cloning of the Full-Length Coding Sequence of SNORF2 (Rat NPFF1)

[0200] Pools of the rat hypothalamic cDNA library “I” were screened byPCR with SNORF2-specific primers JAB208 and JAB209 and the Expand LongTemplate PCR system (Boehringer-Mannheim, Indianapolis, Ind.) with thefollowing PCR protocol: 94° C. hold for 3 minutes; 40 cycles of 94° C.for 1 minute, 68° C. for 2 minutes; 4 minute hold at 68° C.; 4° C. holduntil the samples are run on a gel. This screen yielded a positive poolI36E and a positive sub-pool I36E-17. High stringency hybridization ofisolated colonies from I36E-17 with the SNORF2-specific oligonucleotideprobe JAB211 and subsequent PCR testing of positive colonies indicatedthat the isolated clone I36E-17-1B-1 contained at least a partial cloneof SNORF2. Sequencing of I36E-17-1B-1 revealed that this insertcontained the coding region from the TMIII-TMIV loop through the stopcodon, including some 3′ untranslated sequence. From this sequence, anew forward primer, JAB221, was designed in TMV. PCR screening of asecond rat hypothalamic cDNA library “J” with primers JAB221 and JAB209,and subsequent colony hybridization with the JAB211 probe on a lowcomplexity positive sub-pool resulted in the isolation of a SNORF2 cloneJ-13-16-A1. Full-length double-stranded sequence of SNORF2 wasdetermined by sequencing both strands of the J-13-16-A1 plasmid using anABI 377 sequencer as described above. This insert is about 2.8 kb inlength with an approximately 200 bp 5′ untranslated region, a 1296 bpcoding region, and a 1.3 kb 3′untranslated region. The clone is also inthe correct orientation for expression in the mammalian expressionvector pEXJ.T7. This construct of SNORF2 in pEXJ.T7 was designated BN-6.The full length SNORF2 was determined to be most like the orexin 1receptor (45% DNA identity, 35% amino acid identity), orexin 2 receptor(40% DNA identity, 32% amino acid identity), and NPY2 receptor (47% DNAidentity, 29% amino acid identity), although several other Gprotein-coupled receptors also displayed significant homology. Therewere no sequences in the Genbank databases (genembl, sts, est, gss, orswissprot) that were identical to SNORF2. SNORF2 also showed significanthomology (85% nucleotide identity, 93% amino acid identity) to a partialG protein-coupled receptor fragment in the Synaptic PharmaceuticalCorporation in-house database, designated PLC29b. PLC29b, which includespart of the amino terminus through TMIII, was originally isolated from ahuman genomic library using oligonucleotide probes for NPY4. Subsequentscreening of a human hippocampal cDNA library yielded an overlappingsequence extending into TMIV. Based on sequence similarity, this humansequence appears to be a partial clone of the human homolog of SNORF2.

[0201] The following is a list of primers and their associated sequenceswhich were used in the cloning of these receptors: JAB126:5′-GYNTWYRYNNTNWSNTGGHTNCC-3′ (Seq. ID No. 9) JAB108:5′-AVNADNGBRWAVANNANNGGRTT-3′ (Seq. ID No. 10) JAB1:5′-TTATGCTTCCGGCTCGTATGTTGTG-3′ (Seq. ID No. 11) JAB2:5′-ATGTGCTGCAAGGCGATTAAGTTGGG-3′ (Seq. ID No. 12) JAB208:5′-GGTGCTGCTGCTGCTCATCGACTATG-3′ (Seq. ID No. 13) JAB209:5′-TTGGCGCTGCTGTGGAAGAAGGCCAG-3′ (Seq. ID No. 14) JAB221:5′-CGGTGCTCTTCGCGCACATCTACC-3′ (Seq. ID No. 15) JAB211:5′-TGCCAAGGGGAAGGCGTAGACCGACAGCAGGTGCAGTTGCA (Seq. ID No. 16)GCTCGATCAGCTCCCCATA-3′

[0202] Isolation of the Full-Length Human SNORF2 Receptor Gene (HumanNPFF1)

[0203] The full-length, intronless version of the human NPFF1 receptorgene may be isolated using standard molecular biology techniques andapproaches such as those briefly described below:

[0204] Approach #1:

[0205] To obtain a full-length human NPFF1 receptor, a human cosmidlibrary was screened with a ³²P-labeled oligonucleotide probe, BB609,corresponding to the 2/3 loop of the PLC29b clone. A positive clone wasisolated and partially sequenced, revealing part of the amino terminusand TMs I and II.

[0206] The full-length sequence may be obtained by sequencing thiscosmid clone with additional sequencing primers. Since at least twointrons are present in this gene, one in the amino terminus and one justafter the third transmembrane domain, the full-length intronless genemay be obtained from cDNA using standard molecular biology techniques.For example, a forward PCR primer designed in the 5′UT and a reverse PCRprimer designed in the 3′UT may be used to amplify a full-length,intronless gene from cDNA. RT-PCR localization has identified severalhuman tissues which could be used for this purpose, includingcerebellum, spinal cord, hippocampus, lung and kidney. Standardmolecular biology techniques could be used to subclone this gene into amammalian expression vector.

[0207] Approach #2

[0208] Standard molecular biology techniques could be used to screencommercial human cDNA phage libraries by hybridization under highstringency with a ³²P-labeled oligonucleotide probe, BB609,corresponding to the 2/3 loop of the PLC29b clone. One may isolate afull-length human NPFF1 by obtaining a plaque purified clone from thelambda libraries and then subjecting the clone to direct DNA sequencingusing primers from the PLC29b sequence. Alternatively, standardmolecular biology techniques could be used to screen in-house human cDNAplasmid libraries by PCR amplification of library pools using primers tothe human NPFF1 sequence (BB629, forward primer in TMI, and A71, reverseprimer in TMIV). A full-length clone could be isolated by Southernhybridization of colony lifts of positive pools with a ³²P-labeledoligonucleotide probe, BB609, corresponding to the 2/3 loop of thePLC29b clone.

[0209] Approach #3:

[0210] As yet another alternative method, one could utilize 3′ and 5′RACE to generate PCR products from human cDNA expressing human NPFF1(for example, cerebellum, spinal cord, hippocampus, lung and kidney),which contain the additional sequences of human NPFF1. For 5′ RACE, areverse primer derived from PLC29b between the amino terminus and TM IVcould be used to amplify the additional amino terminus sequence forhNPFF1. For 3′ RACE, a forward primer derived from PLC29b between theamino terminus and TM IV could be used to amplify the additional 3′sequence for hNPFF1, including TMs 5-7 and the COOH terminus. These RACEPCR product could then be sequenced to determine the missing sequence.This new sequence could then be used to design a forward PCR primer inthe 5′UT and a reverse primer in the 3′UT. These primers could then beused to amplify a full-length hNPFF1 clone from human cDNA sources knownto express NPFF1 (for example, cerebellum, spinal cord, hippocampus.lung and kidney). BB609:5′-CCACCCTTGTGGACAACCTCATCACTGGGTGGCCCTTCGACAATGCC (Seq. ID No. 17)ACATGC-3′ BB629: 5′-CTGCTCTGCATGGTGGGCAACACC-3′ (Seq. ID No. 18) A71:5′-GACGGCGATGGTGACGAGCGC-3′ (Seq. ID No. 19)

[0211] Cloning of Human NPFF1 Receptor

[0212] The sequence of the human NPFF1 (hNPFF1) receptor from theinitiating methionine to TMIV was determined to be present in a partialclone, plc29b, found in a Synaptic Pharmaceutical Corporation in-housedatabase. In order to isolate the full-length hNPFF1 receptor cDNA, ahuman cosmid library (Stratagene) was screened with a ³²P-labeled probe(BB609) corresponding to the II/III loop of plc29b. Partial DNAsequencing of one positive clone from this library, COS28a revealedsimilar sequence as had been previously shown for plc29b, with an introndownstream of TMIII. In order to obtain sequence in the 3′ end ofhNPFF1, COS28a was amplified with a vector primer and BB702, BB703 orBB704, forward primers in TMIV. DNA sequencing of these PCR productsresulted in the identification of TMIV through the stop codon.

[0213] Next, an in-house human spinal cord library was screened by PCRusing a forward primer in the region of the initiating methionine(BB729) and a reverse primer corresponding to TMIV (BB728). One positivepool, W4, was subdivided and a positive sub-pool was screened by colonyhybridization with a ³²P-labeled probe from TMII, BB676. Plasmid DNA wasisolated for clone W4-18-4,renamed BO98, and DNA sequencing revealedthat it was full-length but in the wrong orientation for expression inthe expression vector pEXJ. To obtain a full-length hNPFF1 construct inthe correct orientation, BO98 was amplified with BB757, a forward primerat the initiating methionine which contained an upstream BamHI site, andBB758, a reverse primer at the stop codon which contained a EcoRI site.The products from 3 independent PCR reactions were ligated intopcDNA3.1+ and transformed into DH5α cells. The sequence of one of thesetransformants, 3.3, was identical to the hNPFF1 sequence previouslydetermined from the consensus of BO98, COS28a and plc29b. Clone 3.3 wasrenamed BO102.

[0214] The hNPFF1 clone contains an open reading frame with 1293nucleotides and predicts a protein of 430 amino acids (FIGS. 11 and 12).Hydrophobicity analysis reveals seven hydrophobic domains which arepresumed to be transmembrane domains (FIG. 13). The sequence of hNPFF1was determined to be most similar to the rat NPFF1 (86% nucleotideidentity, 87% amino acid identity) and human NPFF2 (56% nucleotideidentity, 49% amino acid identity (FIG. 14)). The human NPFF1 receptoralso shares homology with human orexin₁ (53% nucleotide identity, 35%amino acid identity), human orexin₂ (43% nucleotide identity, 33% aminoacid identity), human NPY₂ (47% nucleotide identity, 31% amino acididentity), human CCK_(A) (46% nucleotide identity, 32% amino acididentity); and human CCK_(B) (46% nucleotide identity, 26% amino acididentity).

[0215] The following primers and probes were used in the cloning ofhNPFF1: BB676: 5′-GTCACCAACATGTTCATCCTCAACCTGGCTGTCAGTGACCTGCT (Seq. IDNo. 20) GGTGGGCATCTTCTGCATGCC-3′ BB702: 5′-GCGAGAAGCTGACCCTGCGGAAGG-3′(Seq. ID No. 21) BB703: 5′-TCGTCACCATCGCCGTCATCTGGG-3′ (Seq. ID No. 22)BB704: 5′-CGTCATCTGGGCCGAGGGACACAG-3′ (Seq. ID No. 23) BB728:5′-TGACGGCGATGGTGACGAGCGCC-3′ (Seq. ID No. 24) BB729:5′-CAGCCTCCCAACAGCAGTTGGCC-3′ (Seq. ID No. 25) BB757:5′-TAGCAAGGATCCGCATATGGAGGGGGAGCCCTCCC-3′ (Seq. ID No. 26) BB758:5′-CTTCATGAATTCATCGCCTGCATGTATCTCGTGTCC- 3′ (Seq. ID No. 27)

[0216] Cloning of Human NPFF2 Receptor

[0217] Discovery of an Expressed Sequence Tag (EST) AA449919 in GENEMBLHomologous to rNPFF1 (hNPFF2)

[0218] A FASTA search of GENEMBL with the full-length sequence of ratNPFF1 (rNPFF1) resulted in the identification of an EST (Accessionnumber AA449919) with a high degree of homology to NPFF1 (57% identityat the DNA level). AA449919 is a 532 bp sequence annotated in Genbank as“Soares total fetus Nb2HF8 9w Homo sapiens cDNA clone 788698 5′ similarto SW:NYR_DROME P25931 NEUROPEPTIDE Y RECEPTOR,” which when translatedcorresponds to the region between the first extracellular loop and thebeginning of the sixth transmembrane domain of rNPFF1. GAP analysis ofAA449919 with rNPFF1 indicated that there is 57% DNA identity and a 50%amino acid identity between the two receptor sequences over this region.AA449919 displays 60% DNA identity and 59% amino acid identity over theregion that overlaps with the known sequence for hNPFF1 (firstextracellular loop to TM4), while over the same range rNPFF1 is 62% and61% identical to AA449919 at the DNA and amino acid levels,respectively. In comparison, hNPFF1 and rNPFF1 share 86% DNA identityand 92% amino acid identity over this region. Given the strong degree ofidentity between AA449919 and rNPFF1, AA449919 was given the nameNPFF-like (hNPFF2).

[0219] Cloning the Full-Length Sequence of (NPFF-Like) hNPFF2

[0220] To determine the full-length coding sequence of AA449919, 5′/3′Rapid Amplification of cDNA ends (RACE) was performed on Clontech HumanSpleen Marathon-Ready cDNA (Clontech, Palo Alto, Calif.). For 5′ RACE, 5μl template (human spleen Marathon-Ready cDNA was amplified witholigonucleotide primers JAB256 and AP1, the Expand Long DNA Template PCRSystem (Boehringer-Mannheim, Indianapolis, Ind.) and the following PCRprotocol were used: 94° C. hold for 3 minutes; 5 cycles of 94° C. for 30seconds, 72° C. for 4 minutes; 5 cycles of 94° C. for 30 seconds, 70° C.for 4 minutes; 30 cycles of 94° C. for 30 seconds, 68° C. for 4 minutes;68° C. hold for 4 minutes; 4° C. hold until products were ready to beloaded on a gel. 1 μl of this reaction was subjected to a second roundof amplification with primers JAB260 and AP2 and the same PCR protocol.For 3′ RACE, 5 μl human spleen Marathon-Ready cDNA was subjected to PCRwith primers JAB257 and AP1 with the same PCR protocol that was used for5′ RACE. 1 μl of this reaction was subjected to another round ofamplification using AP2 and JAB258 and the same PCR conditions.

[0221] The products were run on a 1% agarose TAE gel and bands greaterthan 500 bp were extracted from the gel using the QIAQUICK gelextraction kit (QIAGEN, Chatsworth, Calif.). 5 μl of each purified bandfrom the 5′ and 3′ RACE reactions were directly sequenced with primersJAB261 (5′ products) and JAB259 (3′ products) using the ABI Big Dyecycle sequencing protocol and ABI377 sequencers (ABI, Foster City,Calif.). The Wisconsin Package (GCG, Genetics Computer Group, Madison,Wis.) and Sequencer 3.0 (Gene Codes Corporation, Ann Arbor, Mich.) wereused to put together the full-length contiguous sequence of hNPFF2 fromthe AA449919 EST and the RACE products.

[0222] To attain the full-length hNPFF-like (hNPFF2) coding sequence forexpression, human spinal cord cDNA was amplified in eight independentPCR reactions using the Expand Long Template PCR System with buffer I(four of the eight reactions) or buffer 3 (4 reactions) and twooligonucleotide primers with restriction sites incorporated into their5′ ends: BB675 is a forward primer upstream of the initiating methionineand contains a BamHI site, and BB663. The PCR conditions for thisreaction were as follows: 94° C. hold for 5 minutes; 37 cycles of 94° C.for 30 seconds, 64° C. for 30 seconds, 68° C. for 2 minutes; a 7 minutehold at 68° C., and a 4° C. hold until products were ready to be loadedon a gel. The products were electrophoresed on a 1% agarose TAE gel, anda band of approximately 1.35 kb was cut and purified using the QIAQUICKgel extraction kit. The purified bands of seven of the eight reactionswere cut with BamHI and EcoRI, gel purified again using the same method,and ligated into pcDNA3.1(+) (Invitrogen, Carlsbad, Calif.). Eighteencolonies from the subsequent transformations were picked and determinedto be positive for NPFF-like by PCR. Eight of these 18 clones were fullysequenced, and one of these, BO89, was determined to be a full lengthclone with no point mutations. This construct was renamedpcDNA3.1-hNPFF2b.

[0223] For expression of NPFF-like in oocytes, one ul of each of theseeight ligations of the BB675-BB663 PCR product into pcDNA3.1(+) wassubjected to PCR with AN35, a pcDNA3.1 primer at the CMV promoter site,and the 3′ NPFF-like primer BB663 using the Expand Long Template PCRSystem and the following PCR protocol: 94° C. hold for 3 minutes; 37cycles of 94° C. for 30 seconds, 65° C. for 30 seconds, 68° C. for 2minutes; a 7 minute hold at 68° C., and a 4° C. hold until products wereready for in vitro transcription. Of the seven PCR reactions, sixyielded products of the expected size.

[0224] The following is a list of primers and their associated sequenceswhich were used in the cloning of this receptor (hNPFF2): AN35:5′-CGTGTACGGTGGGAGGTCTATATAAGCAGAG- 3′ (Seq. ID No. 28) AP1:5′-CCATCCTAATACGACTCACTATAGGGC-3′ (Seq. ID No. 29) AP2:5′-ACTCACTATAGGGCTCGAGCGGC-3′ (Seq. ID No. 30) JAB256:5′-TGATAGTGAGCTTTGGTTTAAAAGGG-3′ (Seq. ID No. 31) JAB257:5′-GAAGATCTACACCACTGTGCTGTTTG-3′ (Seq. ID No. 32) JAB258:5′-AACATCTACCTGGCTCCCCTCTCCC-3′ (Seq. ID No. 33) JAB259:5′-TTGTCATCATGTATGGAAGGATTGG-3′ (Seq. ID No. 34) JAB260:5′-GACCACACACTGGAACCTATCTAC-3′ (Seq. ID No. 35) JAB261:5′-GCAATTGCAACTAACGTAAAGACTG-3′ (Seq. ID No. 36) BB675:5′-TAGCAAGGATCCGAGGTTCATCATGAATGAGAAATGG-3′ (Seq. ID No. 37) BB663:5′-CTTCATGAATTCGCGTAGTAGAGTTAGGATTATCAC-3′ (Seq. ID No. 38)

[0225] For expression of NPFF2, mRNA transcripts were generated asdescribed for NPFF1, using PCR products from ligation reactions orlinearized DNA from B089 as DNA templates. Oocytes were injected with5-50 ng NPFF2 mRNA and incubated as previously described.

[0226] Cell Culture

[0227] COS-7 cells are grown on 150 mm plates in DMEM with supplements(Dulbecco's Modified Eagle Medium with 10% bovine calf serum, 4 mMglutamine, 100 units/ml penicillin/100 μg/ml streptomycin) at 37° C., 5%CO₂. Stock plates of COS-7 cells are trypsinized and split 1:6 every 3-4days.

[0228] Human embryonic kidney 293 cells are grown on 150 mm plates inDMEM with supplements (10% bovine calf serum, 4 mM glutamine, 100units/ml penicillin/100 μg/ml streptomycin) at 37° C., 5% CO₂. Stockplates of 293 cells are trypsinized and split 1:6 every 3-4 days.

[0229] Mouse fibroblast LM(tk-) cells are grown on 150 mm plates inD-MEM with supplements (Dulbecco's Modified Eagle Medium with 10% bovinecalf serum, 4 mM glutamine, 100 units/ml penicillin/100 μg/mlstreptomycin) at 37° C., 5% CO₂. Stock plates of LM(tk-) cells aretrypsinized and split 1:10 every 3-4 days.

[0230] Chinese hamster ovary (CHO) cells were grown on 150 mm plates inHAM's F-12 medium with supplements (10% bovine calf serum, 4 mML-glutamine and 100 units/ml penicillin/ 100 ug/ml streptomycin) at 37°C., 5% CO₂. Stock plates of CHO cells are trypsinized and split 1:8every 3-4 days.

[0231] Mouse embryonic fibroblast NIH-3T3 cells are grown on 150 mmplates in Dulbecco's Modified Eagle Medium (DMEM) with supplements (10%bovine calf serum, 4 mM glutamine, 100 units/ml penicillin/100 μg/mlstreptomycin) at 37° C., 5% CO₂. Stock plates of NIH-3T3 cells aretrypsinized and split 1:15 every 3-4 days. Sf9 and Sf21 cells are grownin monolayers on 150 mm tissue culture dishes in TMN-FH mediasupplemented with 10% fetal calf serum, at 27° C., no CO₂. High Fiveinsect cells are grown on 150 mm tissue culture dishes in Ex-Cell 400™medium supplemented with L-Glutamine, also at 27° C., no CO₂.

[0232] Transient Transfection

[0233] Receptors studied may be transiently transfected into COS-7 cellsby the DEAE-dextran method using 1 μg of DNA/10⁶ cells (Cullen, 1987).In addition, Schneider 2 Drosophila cells may be cotransfected withvectors containing the receptor gene under control of a promoter whichis active in insect cells, and a selectable resistance gene, e.g., theG418 resistant neomycin gene, for expression of the polypeptidesdisclosed herein.

[0234] Stable Transfection

[0235] DNA encoding the human receptor disclosed herein may beco-transfected with a G-418 resistant gene into the human embryonickidney 293 cell line by a calcium phosphate transfection method (Cullen,1987). Stably transfected cells are selected with G-418.

[0236] Membrane Preparations

[0237] LM(tk-) cells stably transfected with the DNA encoding the humanreceptor disclosed herein may be routinely converted from an adherentmonolayer to a viable suspension. Adherent cells are harvested withtrypsin at the point of confluence, resuspended in a minimal volume ofcomplete DMEM for a cell count, and further diluted to a concentrationof 106 cells/ml in suspension media (10% bovine calf serum, 10% 10XMedium 199 (Gibco), 9 mM NaHCO₃, 25 mM glucose, 2 mM L-glutamine, 100units/ml penicillin/100 μg/ml streptomycin, and 0.05% methyl cellulose).Cell suspensions are maintained in a shaking incubator at 37° C., 5% CO₂for 24 hours. Membranes harvested from cells grown in this manner may bestored as large, uniform batches in liquid nitrogen. Alternatively,cells may be returned to adherent cell culture in complete DMEM bydistribution into 96-well microtiter plates coated with poly-D-lysine(0.01 mg/ml) followed by incubation at 37° C., 5% CO₂ for 24 hours.

[0238] Generation of Baculovirus

[0239] The coding region of DNA encoding the human receptors disclosedherein may be subcloned into pBlueBacIII into existing restriction sitesor sites engineered into sequences 5′ and 3′ to the coding region of thepolypeptides. To generate baculovirus, 0.5 μg of viral DNA (BaculoGold)and 3 μg of DNA construct encoding a polypeptide may be co-transfectedinto 2×10⁶ Spodoptera frugiperda insect Sf9 cells by the calciumphosphate co-precipitation method, as outlined by Pharmingen (in“Baculovirus Expression Vector System: Procedures and Methods Manual”).The cells then are incubated for 5 days at 27° C.

[0240] The supernatant of the co-transfection plate may be collected bycentrifugation and the recombinant virus plaque purified. The procedureto infect cells with virus, to prepare stocks of virus and to titer thevirus stocks are as described in Pharmingen's manual.

[0241] Radioligand Binding Assays

[0242] Cells may be screened for the presence of endogenous humanreceptor using radioligand binding or functional assays (described indetail in the following experimental description). Cells with either noor a low level of the endogenous human receptors disclosed hereinpresent may be transfected with the human receptors.

[0243] Transfected cells from culture flasks are scraped into 5 ml of 20mM Tris-HCl, 5 mM EDTA, pH 7.5, and lysed by sonication. The celllysates are centrifuged at 1000 rpm for 5 min. at 4° C., and thesupernatant is centrifuged at 30,000× g for 20 min. at 4° C. The pelletis suspended in binding buffer (50 mM Tris-HCl, 60 mM NaCl, 1 mM MgCl,33 μM EDTA, 33 μM EGTA at pH 7.4 supplemented with 0.2% BSA, 2 μg/mlaprotinin, and 20 μM bestatin). Optimal membrane suspension dilutions,defined as the protein concentration required to bind less than 10% ofthe added radioligand, are added to 96-well polpropylene microtiterplates containing ³H-labeled compound, unlabeled compounds, and bindingbuffer to a final volume of 250 μl. In equilibrium saturation bindingassays membrane preparations are incubated in the presence of increasingconcentrations of [³H]-labeled compound. The binding affinities of thedifferent compounds are determined in equilibrium competition bindingassays, using [125I]-labeled compound in the presence of ten to twelvedifferent concentrations of the displacing ligands.

[0244] Competition assay:

[0245] 50 pM radioligand, 10-12 points. Binding reaction mixtures areincubated for 2 hr at 25° C., and the reaction stopped by filtrationthrough a double layer of GF filters treated with 0.1%polyethyleneimine, using a cell harvester. Wash buffer: 50 mM Tris-HCl,0.1% BSA. Radioactivity may be measured by scintillation counting anddata are analyzed by a computerized non-linear regression program.Non-specific binding is defined as the amount of radioactivity remainingafter incubation of membrane protein in the presence of 1 μM finalconcentration unlabeled. Protein concentration may be measured by theBradford method using Bio-Rad Reagent, with bovine serum albumin as astandard.

[0246] Functional Assays

[0247] Cells may be screened for the presence of endogenous mammalianreceptor using radioligand binding or functional assays (described indetail in the above or following experimental description,respectively). Cells with no or a low level of endogenous receptorpresent may be transfected with the mammalian receptor for use in thefollowing functional assays.

[0248] A wide spectrum of assays can be employed to screen for thepresence of receptor ligands. These range from traditional measurementsof phosphatidyl inositol, cAMP, Ca⁺⁺, and K⁺, for example; to systemsmeasuring these same second messengers but which have been modified oradapted to be higher throughput, more generic, and more sensitive; tocell based platforms reporting more general cellular events resultingfrom receptor activation such as metabolic changes, differentiation, andcell division/proliferation, for example; to high level organism assayswhich monitor complex physiological or behavioral changes thought to beinvolved with receptor activation including cardiovascular, analgesic,orexigenic, anxiolytic, and sedation effects, for example.

[0249] Cyclic AMP (cAMP) Formation Assay

[0250] The receptor-mediated inhibition of cyclic AMP (cAMP) formationmay be assayed in transfected cells expressing the mammalian receptors.Cells are plated in 96-well plates and incubated in Dulbecco's phosphatebuffered saline (PBS) supplemented with 10 mM HEPES, 5 mM theophylline,2 μg/ml aprotinin, 0.5 mg/ml leupeptin, and 10 μg/ml phosphoramidon for20 min at 37° C., in 5% CO₂. Test compounds are added and incubated foran additional 10 min at 37° C. The medium is then aspirated and thereaction stopped by the addition of 100 mM HCl. The plates are stored at4° C. for 15 min, and the cAMP content in the stopping solution measuredby radioimmunoassay. Radioactivity may be quantified using a gammacounter equipped with data reduction software.

[0251] Arachidonic Acid Release Assay

[0252] Cells stably transfected with the mammalian receptor are seededinto 96 well plates and grown for 3 days in HAM's F-12 with supplements.³H-arachidonic acid (specific activity=0.75 μCi/ml) is delivered as a100 μL aliquot to each well and samples were incubated at 37° C., 5% CO₂for 18 hours. The labeled cells are washed three times with 200 μL HAM'sF-12. The wells are then filled with medium (200 μL) and the assay isinitiated with the addition of peptides or buffer (22 μL). Cells areincubated for 30 min at 37° C., 5% CO₂. Supernatants are transferred toa microtiter plate and evaporated to dryness at 75° C. in a vacuum oven.Samples are then dissolved and resuspended in 25 μL distilled water.Scintillant (300 μL) is added to each well and samples are counted for³H in a Trilux plate reader. Data are analyzed using nonlinearregression and statistical techniques available in the GraphPAD Prismpackage (San Diego, Calif.).

[0253] Intracellular Calcium Mobilization Assay

[0254] The intracellular free calcium concentration may be measured bymicrospectroflourometry using the fluorescent indicator dye Fura-2/AM(Bush et al, 1991). Stably transfected cells are seeded onto a 35 mmculture dish containing a glass coverslip insert. Cells are washed withHBS and loaded with 100 μL of Fura-2/AM (10 μM) for 20 to 40 min. Afterwashing with HBS to remove the Fura-2/μM solution, cells areequilibrated in HBS for 10 to 20 min. Cells are then visualized underthe 40× objective of a Leitz Fluovert FS microscope and fluorescenceemission is determined at 510 nM with excitation wavelengths alternatingbetween 340 nM and 380 nM. Raw fluorescence data are converted tocalcium concentrations using standard calcium concentration curves andsoftware analysis techniques.

[0255] Phosphoinositide Metabolism Assay

[0256] Cells stably expressing the mammalian receptor cDNA are plated in96-well plates and grown to confluence. The day before the assay thegrowth medium is changed to 100 μl of medium containing 1% serum and 0.5μCi [³H]myo-inositol, and the plates are incubated overnight in a CO₂incubator (5% CO₂ at 37° C.). Alternatively, arachidonic acid releasemay be measured if [³H]arachidonic acid is substituted for the[³H]myo-inositol. Immediately before the assay, the medium is removedand replaced by 200 μL of PBS containing 10 mM LiCl, and the cells areequilibrated with the new medium for 20 min. During this interval cellsare also equilibrated with the antagonist, added as a 10 μL aliquot of a20-fold concentrated solution in PBS. The [³H]inositol-phosphatesaccumulation from inositol phospholipid metabolism may be started byadding 10 μL of a solution containing the agonist. To the first well 10μL may be added to measure basal accumulation, and 11 differentconcentrations of agonist are assayed in the following 11 wells of eachplate row. All assays are performed in duplicate by repeating the sameadditions in two consecutive plate rows. The plates are incubated in aCO₂ incubator for 1 hr. The reaction may be terminated by adding 15 μLof 50% v/v trichloroacetic acid (TCA) followed by a 40 min. incubationat 4° C. After neutralizing TCA with 40 μL of 1 M Tris, the content ofthe wells may be transferred to a Multiscreen HV filter plate(Millipore) containing Dowex AG1-X8 (200-400 mesh, formate form). Thefilter plates are prepared adding 200 μL of Dowex AG1-X8 suspension (50%v/v, water: resin) to each well. The filter plates are placed on avacuum manifold to wash or elute the resin bed. Each well is washed 2times with 200 μL of water, followed by 2×200 μL of 5 mM sodiumtetraborate/60 mM ammonium formate. The [³H]IPs are eluted into empty96-well plates with 200 μL of 1.2 M ammonium formate/0.1 formic acid.The content of the wells is added to 3 ml of scintillation cocktail, andthe radioactivity is determined by liquid scintillation counting.

[0257] GTPγS Functional Assay

[0258] Membranes from cells transfected with the mammalian receptors aresuspended in assay buffer (50 mM Tris, 100 mM NaCl, 5 mM MgCl₂, pH 7.4)supplemented with 0.1% BSA, 0.1% bacitracin and 10 AM GDP. Membranes areincubated on ice for 20 minutes, transferred to a 96-well Milliporemicrotiter GF/C filter plate and mixed with GTPγ³⁵S (e.g., 250,000cpm/sample, specific activity ˜1000 Ci/mmol) plus or minus GTPγS (finalconcentration=100 μM). Final membrane protein concentration≈90 μg/ml.Samples are incubated in the presence or absence of porcine galanin(final concentration=1 μM) for 30 min. at room temperature, thenfiltered on a Millipore vacuum manifold and washed three times with coldassay buffer. Samples collected in the filter plate are treated withscintillant and counted for ³⁵S in a Trilux (Wallac) liquidscintillation counter. It is expected that optimal results are obtainedwhen the mammalian receptor membrane preparation is derived from anappropriately engineered heterologous expression system, i.e., anexpression system resulting in high levels of expression of themammalian receptor and/or expressing G-proteins having high turnoverrates (for the exchange of GDP for GTP). GTPγS assays are well-known inthe art, and it is expected that variations on the method describedabove, such as are described by e.g., Tian et al. (1994) or Lazareno andBirdsall (1993), may be used by one of ordinary skill in the art.

[0259] MAP Kinase Assay

[0260] MAP kinase (mitogen activated kinase) may be monitored toevaluate receptor activation. MAP kinase is activated by multiplepathways in the cell. A primary mode of activation involves theras/raf/MEK/MAP kinase pathway. Growth factor (tyrosine kinase)receptors feed into this pathway via SHC/Grb-2/SOS/ras. Gi coupledreceptors are also known to activate ras and subsequently produce anactivation of MAP kinase. Receptors that activate phospholipase C (Gqand G11) produce diacylglycerol (DAG) as a consequence of phosphatidylinositol hydrolysis. DAG activates protein kinase C which in turnphosphorylates MAP kinase.

[0261] MAP kinase activation can be detected by several approaches. Oneapproach is based on an evaluation of the phosphorylation state, eitherunphosphorylated (inactive) or phosphorylated (active). Thephosphorylated protein has a slower mobility in SDS-PAGE and cantherefore be compared with the unstimulated protein using Westernblotting. Alternatively, antibodies specific for the phosphorylatedprotein are available (New England Biolabs) which can be used to detectan increase in the phosphorylated kinase. In either method, cells arestimulated with the mitogen and then extracted with Laemmli buffer. Thesoluble fraction is applied to an SDS-PAGE gel and proteins aretransferred electrophoretically to nitrocellulose or Immobilon.Immunoreactive bands are detected by standard Western blottingtechnique. Visible or chemiluminescent signals are recorded on film andmay be quantified by densitometry.

[0262] Another approach is based on evaluation of the MAP kinaseactivity via a phosphorylation assay. Cells are stimulated with themitogen and a soluble extract is prepared. The extract is incubated at30° C. for 10 min with gamma-³²P-ATP, an ATP regenerating system, and aspecific substrate for MAP kinase such as phosphorylated heat and acidstable protein regulated by insulin, or PHAS-I. The reaction isterminated by the addition of H₃PO₄ and samples are transferred to ice.An aliquot is spotted onto Whatman P81 chromatography paper, whichretains the phosphorylated protein. The chromatrography paper is washedand counted for ³²P in a liquid scintillation counter. Alternatively,the cell extract is incubated with gamma-³²P-ATP, an ATP regeneratingsystem. and biotinylated myelin basic protein bound by streptavidin to afilter support. The myelin basic protein is a substrate for activatedMAP kinase. The phosphorylation reaction is carried out for 10 min at30° C. The extract can then by aspirated through the filter, whichretains the phosphorylated myelin basic protein. The filter is washedand counted for ³²P by liquid scintillation counting.

[0263] Cell Proliferation Assay

[0264] Receptor activation of a G protein coupled receptor may lead to amitogenic or proliferative response which can be monitored via³H-thymidine uptake. When cultured cells are incubated with³H-thymidine, the thymidine translocates into the nuclei where it isphosphorylated to thymidine triphosphate. The nucleotide triphosphate isthen incorporated into the cellular DNA at a rate that is proportionalto the rate of cell growth. Typically, cells are grown in culture for1-3 days. Cells are forced into quiescence by the removal of serum for24 hrs. A mitogenic agent is then added to the media. 24 hrs later, thecells are incubated with ³H-thymidine at specific activities rangingfrom 1 to 10 uCi/ml for 2-6 hrs. Harvesting procedures may involvetrypsinization and trapping of cells by filtration over GF/C filterswith or without a prior incubation in TCA to extract soluble thymidine.The filters are processed with scintillant and counted for ³H by liquidscintillation counting. Alternatively, adherant cells are fixed in MeOHor TCA, washed in water, and solubilized in 0.05% deoxycholate/0.1 NNaOH. The soluble extract is transferred to scintillation vials andcounted for ³H by liquid scintillation counting.

[0265] Promiscuous Second Messenger Assays

[0266] It is possible to coax receptors of different functional classesto signal through a pre-selected pathway through the use of promiscuousG_(α) subunits. For example, by providing a cell based recetpor assaysystem with an endogenously supplied promiscuous G subunit such asG_(α16) or a chimeric G_(α) subunit such as G_(αzq), a GPCR, which mightnormally prefer to couple through a specific signaling pathway (e.g.,G_(g), G_(i), G_(q), G_(o), etc.), can be made to couple through thepathway defined by the promiscuous G_(α) subunit and upon agonistactivation produce the second messenger associated with that subunit'spathway. In the case of G_(α16) and/or G_(αqz) this would involveactivation of the G_(q) pathway and production of the second messengerphosphotidyl inositol. Through the use of similar strategies and tools,it is possible to bias receptor signaling through pathways producingother second messengers such as Ca⁺⁺, cAMP, and K⁺ currents, forexample.

[0267] Microphysiometric Measurement of Receptor Mediated ExtracellularAcidification Rates

[0268] Because cellular metabolism is intricately involved in a broadrange of cellular events (including receptor activation of multiplemessenger pathways), the use of microphysiometric measurements of cellmetabolism can in principle provide a generic assay of cellular activityarising from the activation of any receptor regardless of the specificsof the receptor's signaling pathway.

[0269] General guidelines for transient receptor expression, cellpreparation and microphysiometric recording are described elsewhere(Salon, J. A. and Owicki, J. A., 1996). Receptors and/or control vectorsare transiently expressed in CHO-K1 cells, by liposome mediatedtransfection according to the manufacturers recommendations (LipofectAMINE, GibcoBRL, Gaithersburg, Md.), and maintained in Ham's F-12complete (10% serum). A total of 10 μg of DNA is used to transfect each75 cm² flask which had been split 24 hours prior to the transfection andjudged to be 70-80% confluent at the time of transfection. 24 hours posttransfection, the cells are harvested and 3×10⁵ cells seeded intomicrophysiometet capsules. Cells are allowed to attach to the capsulemembrane for an additional 24 hours; during the last 16 hours, the cellsare switched to serum-free F-12 complete to minimize ill-definedmetabolic stimulation caused by assorted serum factors. On the day ofthe experiment the cell capsules are transferred to the microphysiometerand allowed to equilibrate in recording media (low buffer RPMI 1640, nobicarbonate, no serum (Molecular Devices Corporation, Sunnyvale, Calif.)containing 0.1% fatty acid free BSA), during which a baselinemeasurement of basal metabolic activity is established.

[0270] A standard recording protocol specifies a 100 μl/min flow rate,with a 2 min total pump cycle which includes a 30 sec flow interrruptionduring which the acidification rate measurement is taken. Ligandchallenges involve a 1 min 20 sec exposure to the sample just prior tothe first post challenge rate measurement being taken, followed by twoadditional pump cycles for a total of 5 min 20 sec sample exposure.Typically, drugs in a primary screen are presented to the cells at 10 μMfinal concentration. Follow up experiments to examine dose-dependency ofactive compounds is then done by sequentially challenging he cells witha drug concentration range that exceeds the amount needed to generateresponses ranging from threshold to maximal levels. Peptides included inthe microphysiometric screen included rat NPFF (FLFQPQRF-NH2) and ratA-18-F-amide (AGEGLSSPFWSLAAPQRF-NH2). Ligand samples are then washedout and the acidification rates reported are expressed as a percentageincrease of the peak response over the baseline rate observed just priorto challenge.

[0271] Receptor/G Protein Co-Transfection Studies

[0272] A strategy for determining whether NPFF can couple preferentiallyto selected G proteins involves co-transfection of NPFF receptor cDNAinto a host cell together with the cDNA for a G protein alpha sub-unit.Examples of G alpha sub-units include members of the Gαai/Gαo class(including Gαt2 and Gαz), the Gαq class, the Gαs class, and the Gα12/13class. A typical procedure involves transient transfection into a hostcell such as COS-7. Other host cells may be used. A key consideration iswhether the cell has a downstream effector (a particular adenylatecyclase, phospholipase C, or channel isoform, for example) to support afunctional response through the G protein under investigation. G proteinbeta gamma sub-units native to the cell are presumed to complete the Gprotein heterotrimer; otherwise specific beta and gamma sub-units may beco-transfected as well. Additionally, any individual or combination ofalpha, beta, or gamma subunits may be co-transfected to optimize thefunctional signal mediated by the receptor.

[0273] The receptor/G alpha co-transfected cells are evaluated in abinding assay, in which case the radioligand binding may be enhanced bythe presence of the optimal G protein coupling or in a functional assaydesigned to test the receptor/G protein hypothesis. In one example, theNPFF receptor may be hypothesized to inhibit cAMP accumulation throughcoupling with G alpha sub-units of the Gαi/Gαo class. Host cellsco-transfected with the NPFF receptor and appropriate G alpha sub-unitcDNA are stimulated with forskolin+/−NPFF agonist, as described above incAMP methods. Intracellular cAMP is extracted for analysis byradioimmunoassay. Other assays may be substituted for cAMP inhibition,including GTPγ³⁵S binding assays and inositol phosphate hydrolysisassays. Host cells transfected with NPFF minus G alpha or with G alphaminus NPFF would be tested simutaneously as negative controls. NPFFreceptor expression in transfected cells may be confirmed in ¹²⁵I-NPFFprotein binding studies using membranes from transfected cells. G alphaexpression in transfected cells may be confirmed by Western blotanalysis of membranes from transfected cells, using antibodies specificfor the G protein of interest.

[0274] The efficiency of the transient transfection procedure is acritical factor for signal to noise in an inhibitory assay, much more sothan in a stimulatory assay. If a positive signal present in all cells(such as forskolin-stimulated cAMP accumulation) is inhibited only inthe fraction of cells successfully transfected with receptor and Galpha, the signal to noise ratio will be poor. One method for improvingthe signal to noise ratio is to create a stably transfected cell line inwhich 100% of the cells express both the receptor and the G alphasubunit. Another method involves transient co-transfection with a thirdcDNA for a G protein-coupled receptor which positively regulates thesignal which is to be inhibited. If the co-transfected cellssimultaneously express the stimulatory receptor, the inhibitoryreceptor, and a requisite G protein for the inhibitory receptor, then apositive signal may be elevated selectively in transfected cells using areceptor-specific agonist. An example involves co-transfection of COS-7cells with 5-HT4, NPFF1, and a G alpha sub-unit. Transfected cells arestimulated with a 5-HT4 agonist+/−NPFF1 protein. Cyclic AMP is expectedto be elevated only in the cells also expressing NPFF1 and the G alphasubunit of interest, and a NPFF-dependent inhibition may be measuredwith an improved signal to noise ratio.

[0275] It is to be understood that the cell lines described herein aremerely illustrative of the methods used to evaluate the binding andfunction of the mammalian receptors of the present invention, and thatother suitable cells may be used in the assays described herein.

[0276] ElectroPhysiology

[0277] Methods for Recording Currents in Xenopus Oocytes

[0278] Oocytes were harvested from Xenopus laevis and injected with mRNAtranscripts as previously described (Quick and Lester, 1994; Smith etal., 1997). NPFF receptors and Gα_(q/z) chimera synthetic RNAtranscripts were synthesized using the T7 polymerase (“Message Machine,”Ambion) from linearized plasmids or PCR products containing the completecoding region of the genes. Oocytes were injected with 10 ng NPFFreceptors synthetic RNA and incubated for 3-8 days at 17 degrees. Threeto eight hours prior to recording, oocytes were injected with 500 pgGα_(q/z) mRNA in order to observe coupling to Ca⁺⁺ activated Cl⁻currents. Dual electrode voltage clamp (Axon Instruments Inc.) wasperformed using 3 M KCl-filled glass microelectrodes having resistancesof 1-2 Mohm. Unless otherwise specified, oocytes were voltage clamped ata holding potential of −80 mV. During recordings, oocytes were bathed incontinuously flowing (1-3 ml/min) medium containing 96 mM NaCl, 2 mMKCl, 1.8 mM CaCl₂, 1 mM MgCl₂, and 5 mM HEPES, pH 7.5 (ND96). Drugs wereapplied either by local perfusion from a 10 ml glass capillary tubefixed at a distance of 0.5 mm from the oocyte, or by switching from aseries of gravity fed perfusion lines.

[0279] Other oocytes may be injected with a mixture of receptor mRNAsand synthetic mRNA encoding the genes for G-protein-activated inwardrectifiers (GIRK1 and GIRK4, U.S. Pat. Nos. 5,734,021 and 5,728,535).Genes encoding G-protein inwardly rectifying K⁺ (GIRK) channels 1 and 4(GIRK1 and GIRK4) may be obtained by PCR using the published sequences(Kubo et al., 1993; Dascal et al., 1993; Krapivinsky et al., 1995 and1995b) to derive appropriate 5′ and 3′ primers. Human heart cDNA may beused as template together with appropriate primers.

[0280] Heterologous expression of GPCRs in Xenopus oocytes has beenwidely used to determine the identity of signaling pathways activated byagonist stimulation (Gundersen et al., 1983; Takahashi et al., 1987).Activation of the phospholipase C (PLC) pathway is assayed by applyingtest compound in ND96 solution to oocytes previously injected with mRNAfor the mammalian receptor and observing inward currents at a holdingpotential of −80 mV. The appearance of currents that reverse at −25 mVand display other properties of the Ca⁺⁺-activated Cl⁻ (chloride)channel is indicative of mammalian receptor-activation of PLC andrelease of IP3 and intracellular Ca⁺⁺. Such activity is exhibited byGPCRs that couple to G_(q).

[0281] Measurement of inwardly rectifying K⁺ (potassium) channel (GIRK)activity may be monitored in oocytes that have been co-injected withmRNAs encoding the mammalian receptor, GIRK1, and GIRK4. The two GIRKgene products co-assemble to form a G-protein activated potassiumchannel known to be activated (i.e., stimulated) by a number of GPCRsthat couple to G_(i) or G_(o) (Kubo et al., 1993; Dascal et al., 1993).Oocytes expressing the mammalian receptor plus the two GIRK subunits aretested for test compound responsivity by measuring K⁺ currents inelevated ⁺K solution containing 49 mM K⁺. Activation of inwardlyrectifying currents that are sensitive to 300 μM Ba⁺⁺ signifies themammalian receptor coupling to a G_(i) or G_(o) pathway in the oocytes.

[0282] Localization of mRNA Coding for Rat NPFF1 Receptors

[0283] Development of Probes for NPFF1:

[0284] To facilitate the production of radiolabeled, antisense RNAprobes a fragment of the gene encoding rat NPFF1 was subcloned into aplasmid vector containing RNA polymerase promotor sites. The full lengthcDNA encoding the rat NPFF1 was digested with Sph I (nucleotides766-1111), and this 345 nucleotide fragment was cloned into the Sph Isite of pGEM 3z, containing both sp6 and T7 RNA polymerase promotorsites. The construct was sequenced to confirm sequence identity andorientation. To synthesize antisense strands of RNA, this construct waslinearized with Hind III and T7 RNA polymerase was used to incorporateradiolabeled nucleotide as described below.

[0285] A probe coding for the rat glyceraldehyde 3-phosphatedehydrogenase (GAPDH) gene, a constitutively expressed protein, was usedconcurrently. GAPDH is expressed at a relatively constant level in mosttissue and its detection is used to compare expression levels of the ratNPFF1 receptors genes in different regions.

[0286] Synthesis of Probes:

[0287] NPFF1 and GAPDH cDNA sequences preceded by phage polymerasepromoter sequences were used to synthesize radiolabeled riboprobes.Conditions for the synthesis of riboprobes were: 0.25-1.0 μg linearizedDNA plasmid template, 1.5 μl of ATP, GTP, UTP (10 mM each) 3 μldithiothreitol (0.1M), 30 units RNAsin RNAse inhibitor, 0.5-1.0 μl(15-20 units/μl) RNA polymerase, 7.0 μl transcription buffer (PromegaCorp.), and 12.5 μl α³²P-CTP (specific activity 3,000 Ci/mmol). 0.1 mMCTP (0.02-1.0 μl) was added to the reactions, and the volumes wereadjusted to 35 μl with DEPC-treated water. Labelina reactions wereincubated at 37° C. for 60 minutes, after which 3 units of RQ1RNAse-free DNAse (Promega Corp.) were added to digest the template.Riboprobes were separated from unincorporated nucleotides usingMicrospin S-300 columns (Pharmacia Biotech). TCA precipitation andliquid scintillation spectrometry were used to measure the amount oflabel incorporated into the probe. A fraction of all riboprobessynthesized was size-fractionated on 0.25 mm thick 7M urea, 4.5%acrylamide sequencing gels. These gels were apposed to screens and theautoradiograph scanned using a phosphorimager (Molecular Dynamics) toconfirm that the probes synthesized were full-length and not degraded.

[0288] Solution Hybridization/Ribonuclease Protection Assay (RPA):

[0289] For solution hybridization 2.0 μg of mRNA isolated from tissueswere used. Negative controls consisted of 30 μg transfer RNA (tRNA) orno tissue blanks. All mRNA samples were placed in 1.5 ml microfuge tubesand vacuum dried. Hybridization buffer (40 μl of 400 mM NaCl, 20 mMTris, pH 6.4, 2 mM EDTA, in 80% formamide) containing 0.25-2.0 E⁶ countsof each probe were added to each tube. Samples were heated at 95° C. for15 min, after which the temperature was lowered to 55° C. forhybridization.

[0290] After hybridization for 14-18 hr, the RNA/probe mixtures weredigested with RNAse A (Sigma) and RNAse T1 (Life Technologies). Amixture of 2.0 μg RNAse A and 1000 units of RNAse T1 in a buffercontaining 330 mM NACl, 10 mM Tris (pH 8.0) and 5 mM EDTA (400 μl) wasadded to each sample and incubated for 90 min at room temperature. Afterdigestion with RNAses, 20 μl of 10% SDS and 50 μg proteinase K wereadded to each tube and incubated at 37° C. for 15 min. Samples wereextracted with phenol/chloroform:isoamyl alcohol and precipitated in 2volumes of ethanol for 1 hr at −70° C. Pellet Paint (Novagen) was addedto each tube (2.0 μg) as a carrier to facilitate precipitation.Following precipitation, samples were centrifuged, washed with cold 70%ethanol, and vacuum dried. Samples were dissolved in formamide loadingbuffer and size-fractionated on a urea/acrylamide sequencing gel (7.0 Murea, 4.5% acrylamide in Tris-borate-EDTA). Gels were dried and apposedto storage phosphor screens and scanned using a phosphorimager(Molecular Dynamics, Sunnydale, Calif.).

[0291] RT-PCR

[0292] For the detection of of low levels of RNA encoding rat NPFF1,RT-PCR was carried out on mRNA extracted from rat tissue. Reversetranscription and PCR reactions were carried out in 50 μl volumes usingEzrTth DNA polymerase (Perkin Elmer). Primers with the followingsequences were used:

[0293] RA Rsnorf2/NPFF F1:

[0294] CTCCTACTACCAACACTCCTCTCC (Seq. ID No. 39)

[0295] RA RSNORF2/NPFF1 B1:

[0296] ACGGGTTACGAGCATCCAG (Seq. ID No. 40)

[0297] These primers will amplify 490 base pair fragment from nucleotide574 to 1064.

[0298] Each reaction contained 0.2 μg mRNA and 0.3 μM of each primer.Concentrations of reagents in each reaction were: 300 μM each of dGTP,DATP, dCTP, dTTP; 2.5 mM Mn(OAc)₂; 50M Bicine; 115 mM K acetate, 8%glycerol and 5 units EzrTth DNA polymerase. All reagents for PCR (exceptmRNA and oligonucleotide primers) were obtained from Perkin Elmer.Reactions were carried out under the following conditions: 65° C., 60min; 94° C., 2 min; (94° C., 1 min; 65° C., 1 min) 35 cycles, 72° C., 10min. PCR reactions were size fractionated by agarose gel electrophoresisusing 10% polyacrylamide. DNA was stained with SYBR Green I (MolecularProbes, Eugene, Oreg.) and scanned on a Molecular Dynamics (Sunnyvale,Calif.) Storm 860 in blue flourescence mode at 450 nM.

[0299] Positive controls for PCR reactions consisted of amplification ofthe target sequence from a plasmid construct, as well as reversetranscribing and amplifying a known sequence. Negative controlsconsisted of mRNA blanks as well as primer blanks. To confirm that themRNA was not contaminated with genomic RNA, samples were digested withRNAses before reverse transcription. Integrity of RNA was assessed byamplification of mRNA coding for GAPDH.

[0300] Localization of mRNA Coding for NPFF-Like Receptors (hNPFF2)Using RT-PCR

[0301] For the detection of low levels of RNA encoding hNPFF2 RT-PCR wascarried out on mRNA extracted from tissue. Reverse transcription and PCRreactions were carried out in 50 μl volumes using EzrTh DNA polymerase(Perkin Elmer). Primers with the following sequences were used: JB 249:5′-GATCAGTGGATTGGTCCAGGGAATATC-3′ (SEQ. ID No. 41) JB 250:5′-CCAGGTAGATGTTGGCAAACAGCAC-3′ (SEQ. ID No. 42).

[0302] These primers will amplify a 332 base pair fragment from TMIII toTMV.

[0303] Each reaction contained 0.1 ug mRNA and 0.3 uM of each primer.Concentrations of reagents in each reaction were 300 uM each of dGTP,DATP, dCTP, dTTP, 2.5 mM Mn(OAc)2, 50 mM Bicine, 115 mM potassiumacetate, 8% glycerol and 5 units EzrTth DNA polymerase. All reagents forPCR (except mRNA and oligonucleotide primers) were obtained from PerkinElmer. Reactions were carried out under the following conditions: 65° C.60 min., 94° C. 2 min, (94° C., 1 min, 65° C. 1 min) 35 cycles, 72° C.10 min. PCR reactions were size fractionated by gel electrophoresisusing 10% polyacrylamide. DNA was stained with SYBR Green I (MolecularProbes, Eugene Oreg.) and scanned on a Molecular Dynamics (Sunnyvale,Calif.) Strom 860 in blue fluorescence mode at 450 nm.

[0304] Positive controls for PCR reactions consisted of amplification ofthe target sequence from a plasmid construct, as well as reversetranscribing and amplifying a known sequence. Negative controlsconsisted of mRNA blanks and primer blanks. To confirm that the mRNA wasnot contaminated with genomic DNA, samples were digested with RNAsesbefore reverse transcription. Integrity of RNA was assessed byamplification of mRNA coding for GAPDH.

[0305] Results and Discussion

[0306] Cloning and Sequencing

[0307] rNPFF1 and hNPFF1

[0308] 100 ng genomic DNA was subjected to MOPAC PCR with two degenerateprimers designed based on the sixth and seventh transmembrane domains ofover 180 receptors from the rhodopsin superfamily of G protein-coupledreceptors. Two products from this reaction, MPR3-RGEN-31 andMPR3-RGEN-45 were found to be identical clones of a novel DNA sequencenot found in the Genbank databases (Genembl, STS, EST, GSS), which had30-40% amino acid identity with the known receptors dopamine D2, orexin1, GALR1, angiotensin 1B and 5HT-2b. This novel clone was given the nameSNORF2.

[0309] The full-length SNORF2 sequence was acquired by screening rathypothalamic cDNA libraries by PCR using specific SNORF2 oligonucleotideprimers. Pools of the rat hypothalamic cDNA library “I” were screened byPCR with SNORF2-specific primers JAB208 and JAB209. This screen yieldeda positive pool I36. Successive PCR screening of sub-pools of this poolfollowed by high stringency hybridization of isolated colonies from thepositive sub-pool I36-17 with the SNORF2-specific oligonucleotide probeindicated that the isolated clone I36E-17-1B-1 contained at least apartial clone of SNORF2. Sequencing of I36E-17-1B-1 revealed that thisinsert contained the coding region from the TMIII-TMIV loop through thestop codon, including some 3′ untranslated sequence. From this sequence,a new forward primer, JAB221, was designed in TMV. PCR screening of asecond rat hypothalamic cDNA library “J” with primers JAB221 and JAB209,and subsequent colony hybridization with the JAB211 probe on a lowcomplexity positive sub-pool resulted in the isolation of a SNORF2 cloneJ-13-16-A1. This clone contained the full-length coding sequence ofSNORF2 (1296 bp) with approximately 200 bp 5′untranslated sequence and1.3 kb 3′ untranslated sequence. The nucleotide sequence of SNORF2 andits translated amino acid sequence are represented in FIGS. 1 and 2,respectively. As shown in FIG. 1, SNORF2 contains two potentialinitiating methionines upstream of TMI.

[0310] Hydophobicity (Kyte-Doolittle) analysis of the amino acidsequence of the full-length clone indicates the presence of sevenhydrophobic regions, which is consistent with the seven transmembranedomains of a G protein-coupled receptor. The seven expectedtransmembrane domains are mapped cut in FIG. 3. A comparison ofnucleotide and peptide sequences of SNORF2 with sequences contained inthe Genbank/EMBL/SwissProtPlus databases reveals that the amino acidsequence of this clone is most related to the orexin 1 and 2 receptors(45% and 40% identity, respectively) as well as the neuropeptide Yreceptors Y1, Y2 and Y4 (˜30% identity). Further homology analysis ofSNORF2 against the Synaptic Pharmaceutical Corporation in-house databaserevealed that SNORF2 has a very high degree of identity with aproprietary Synaptic Pharmaceutical Corporation human partial GPCR clonenamed PLC29b (85% nucleotide identity, 93% amino acid identity). PLC29bwas originally isolated from a human genomic library usingoligonucleotide probes for NPY4, and includes part of the amino terminusand TMs I to IV. Partial nucleotide and amino acid sequence of PLC29b(human SNORF2) is represented in FIGS. 4 and 5, respectively. Based onsequence similarity, PLC29b appears to be a partial clone of the humanhomologue of SNORF2. Therefore, this human homolog of SNORF2 has beennamed hNPFF1. A GAP alignment demonstrating the high homology betweenthese species homologues is represented in FIG. 6.

[0311] SNORF2 has several potential protein kinase C (PKC)phosphorylation motifs throughout its amino acid sequence: threonine 154in the second intracellular loop, threonine 263 and serine 264 in thethird intracellular loop, and serine 363 in the intracellularcarboxy-terminal tail. It also has four potential N-linked glycosylationsites at asparagines 10 and 18 in the amino-terminal tail and atasparagines 113 and 195 in the first and second extracellular loops,respectively.

[0312] hNPFF2

[0313] In analyzing the sequence of rNPFF1 and its homology to othersequences in GenBank, a 532 bp EST with the accession number AA449919was identified which had a high degree of identity to rNPFF1. Thetranslation of this sequence indicated that it coded for the regionbetween the first extracellular loop and the beginning of the sixthtransmembrane domain of a G protein-coupled receptor (GPCR). AlthoughAA449919 was documented as being similar to the Drosophila melanogasterNPY receptor (accession number P25931), it was found that the amino acidsequence encoded by this EST was much more similar to NPFF1. Thepredicted amino acid sequence of AA449919 and rNPFF1 are 50% identical,while the amino acid sequence of the Drosophila NPY receptor is only 31%identical to the translation of AA449919. Because of the high degree ofidentity between AA449919 and rNPFF1, AA449919 was given the namehNPFF2, representing a member of a novel family of NPFF receptors ofwhich there is currently only one member, NPFF1.

[0314] The full length sequence of NPFF-like (hNPFF2) was acquired by5′/3′ RACE using human spleen cDNA as a template, as described above,demonstrating that the coding region of hNPFF-like (hNPFF2) is 1260 bp,coding for a protein of 420 amino acids. Sequencing of clones fromseveral independent PCR reactions using spleen, heart, and spinal cordcDNA as templates and subsequent alignment of these clones withSequencher 3.0 was used to confirm the sequence of hNPFF-like (hNPFF2).The full-length nucleotide sequence of human NPFF2 is shown in FIG. 7,and its translated amino acid sequence is shown in FIG. 8. The sevenputative transmembrane domains of hNPFF-like (hNPFF2) are defined inFIG. 9.

[0315] Like the original EST AA449919, the amino acid sequence encodedby the full-length DNA sequence of hNPFF2 is most similar to rNPFF1 (48%identity), as shown in the GAP alignment between the two receptors inFIG. 10. The next-best matches in SWPLUS to full-length hNPFF2 are theDrosophila NPYR (accession number P25931, 34% identity) and TLR2(accession number P30975, 32% identity), human orexin 1 and 2 receptors(O43613, 31% and O43614, 29%, respectively) and human NPY1 and Y4receptors (P25929, 31% and P50391, 32%, respectively). A Blast search ofthe EST database using the full-length nucleotide sequence of hNPFF2revealed an EST (Accession number AA449920) that is identical to hNPFF2from the end of TM7 through the stop codon. ESTs AA44919 and AA44920 arethe same clone sequenced from 5′ end or the 3′ end, respectively.

[0316] hNPFF2 contains several potential N-linked glycosylation sites.The first three sites, asparagines 8, 20, and 31 are in the N-terminalextracellular domain. Another potential N-linked glycosylation site, atposition 198, is in the second extracellular loop. This receptor alsocontains one potential PKC phosphorylation site at threonine 156 in thesecond intracellular loop, and two potential PKC phosphorylation sitesin the third intracellular loop at threonine 254 and serine 266.

[0317] hNPFF1

[0318] The sequence of hNPFF1 from the initiating methionine to TMIV wasdetermined to be present in a partial clone, plc29b, found in a SynapticPharmaceutical Corporation in-house database. Additional sequence,including TMIV through the stop codon, was determined by sequencing avector-anchored PCR product from a human cosmid library clone identifiedby hybridization with a ³²P-labeled probe (BB609) corresponding to theII/III loop of plc29b. Next, a human spinal cord library was screened byPCR using primers designed against the partial hNPFF1 sequence, BB729and BB728. One positive pool, W4, was subdivided and a positive sub-poolwas screened by colony hybridization with a ³²P-labeled probe from TMIIof hNPFF1, BB676. Plasmid DNA was isolated for clone W4-18-4, renamedBO98, and DNA sequencing revealed that it was full-length but in thewrong orientation for expression in the expression vector pEXJ. Toobtain a full-length hNPFF1 construct in the correct orientation. BO98was amplified with BB757 and BB758, and the resulting product ligatedinto pcDNA3.1 and transformed into DH5α cells. The sequence of one ofthese transformants was identical to the hNPFF1 sequence previouslydetermined from the consensus of BO98 and the two cosmid clones. Thishuman NPFF1 construct in pcDNA3.1 in the correct orientation was renamedBO102.

[0319] The hNPFF1 clone contains an open reading frame with 1293nucleotides and predicts a protein of 430 amino acids (FIGS. 11 and 12).Seven transmembrane domains predicted by hydrophobicity analysis areindicated in FIG. 13. The sequence of hNPFF1 was determined to be mostsimilar to the rat NPFF1 (86% nucleotide identity, 87% amino acididentity) and human NPFF2 (56% nucleotide identity, 49% amino acididentity (FIG. 14)). The human NPFF1 receptor also shares homology withhuman orexin₁ (53% nucleotide identity, 35% amino acid identity), humanorexin2 (43% nucleotide identity, 33% amino acid identity), human NPY₂(47% nucleotide identity, 31% amino acid identity), human CCK_(A) (46%nucleotide identity, 32% amino acid identity), and human CCK_(B) (46%nucleotide identity, 26% amino acid identity)

[0320] Electrophysiology

[0321] NPFF1

[0322] Oocytes injected with both SNORF2 and chimeric Gα_(q/z) syntheticRNAs generated robust inward currents in response to NPFF and therelated peptide A-18-F-amide at 1 μM (FIG. 15A, B). Control oocytesreceiving only G-protein synthetic RNA were unresponsive to thesepeptides. Responses to NPFF were concentration-dependent with athreshold for activation of inward current at 30 nM. The C-terminaltetrapeptide PQRF-amide also elicited responses at a concentration of 10μM (FIG. 15C). Analogs of NPFF containing a tyrosine residue at theN-terminus or internally including Y-8-F-amide, [tyr⁹]A-18-F-amide andY-18-F-amide also displayed activity at 1 μM. Unrelated neuropeptidesand other neurotransmitters including melanin concentrating hormone,orexin B, PYY, 5-HT, nociceptin, galanin and CCK failed to activateoocytes injected with the SNORF2 synthetic RNA. The functionalresponsiveness to NPFF and related peptides strongly suggests thatSNORF2 encodes a receptor for neuropeptide FF (NPFF); therefore SNORF2was renamed NPFF1. Similarly, SNORF2-like was renamed NPFF-like.

[0323] Oocytes injected with NPFF1 and not the chimeric G-proteinsynthetic RNA failed to generated responses to NPFF. This observationsupports the hypothesis that NPFF1 couples to G-proteins of theGα_(i)/Gα_(o)/Gα_(z) class, and by virtue of the N-terminal portion ofGα_(q/z), subsequently activates phospholipase C. In oocytes expressingboth NPFF1 and Gα_(q/z), Cl⁻ currents were abolished by prior injectionof 10 mM EGTA, demonstrating the Ca⁺⁺ dependence of these currents.

[0324] NPFF2

[0325] Oocytes injected with both the NPFF-like PCR product and chimericGα_(q/z) synthetic RNAs generated large inward currents in response to 1μM NPFF (FIG. 16A). A-18-F-amide and PQRF-amide also at 1 μM activatedsimilar inward currents, although the magnitude of currents generated byPGRF-amide were smaller. No activity was observed using FMRF-amide at 1μM. The unrelated neuropeptides orexin A, NPY, galanin, and neurokinin Aat 1 μM also failed to activate responses in oocytes injected withNPFF-like mRNA (FIG. 16B). Oocytes injected with both the NPFF-likeplasmid (B089) and chimeric Gα_(q/z) synthetic RNAs also produced robustcurrents in response to NPFF (FIG. 16C). Based on these results,NPFF-like was renamed NPFF2. Oocytes injected with NPFF2 and notchimeric G-protein mRNA failed to generate responses to NPFF. Thisobservation supports the hypothesis that NPFF2 couples to G-proteins ofthe Gα_(i)/Gα_(o)/Gα_(z) class, and by virtue of the N-terminal portionof Gα_(q/z) subsequently activates phospholipase C.

[0326] Microphysiometry

[0327] CHO cells transiently expressing either NPFF1 alone or NPFF1accompanied by the chimeric protein Gq/Gz produced robust increases inmetabolism when exposed to either NPFF or the related peptideA-18-F-amide as evidenced by increased rates of extracellularacidification when measured by the microphysiometric technique (FIGS.17A and 17B). Whereas control cells, not expressing NPFF1, produced noincrease in acidification rates to either NPFF or A-18-F-amide. In allcases the NPFF1 mediated responses were dose-dependent. CHO cellstransfected with NPFF1 alone produced an EC50 value of 19.3 nM for NPFFwhile cells transfected NPFF1 and the chimeric Gz/Gq produced an EC50 of27.7 nM for NPFF. Challenges with A-18-F-amide were conducted only oncells that had been transfected with NPFF1 alone. These cells producedan EC50 value of 150 nM for A-18-F-amide. The functional responsivenessto NPFF and A-18-F-amide supports the notion that NPFF1 encodes areceptor for neuropeptide FF (NPFF).

[0328] Radioligand Binding Assays

[0329] Cos-7 cells transiently expressing the gene encoding the novelrat NPFF1 receptor were used for pharmacological evaluation. Membranesharvested from transiently transfected Cos-7 cells exhibited highaffinity, saturable [¹²⁵I]D-Tyr-NPFF ([D-Tyr¹ (NMe)Phe³]NPFF) binding.Nonlinear analysis of [¹²⁵I]D-Tyr-NPFF saturation data yielded anequilibrium dissociation constant (K_(d)) of 0.335±0.045 nM and abinding site density (B_(max)) of 180±11 fmol/mg protein. Specific[¹²⁵I]D-Tyr-NPFF binding was about 50% of total binding at a radioligandconcentration equal to the K_(d) value. Mock-transfected host cells didnot display specific [125I]D-Tyr-NPFF binding.

[0330] To further assess the pharmacological identity of the newlyisolated NPFF1 receptor gene, detailed binding properties of clonedNPFF1 receptor were determined from nonlinear analysis of competition ofhigh affinity [¹²⁵I]D-Tyr-NPFF binding. The rank order of affinity ofcompounds to displace specific [¹²⁵I]D-Tyr-NPFF binding is shown inTable 1.

[0331] The binding profile of rat NPFF1 was compared to that of ratspinal cord membranes. Interestingly some differences were observed inthe pharmacological profile between the two preparations. (See Table 2).Notably, frog NPFF did not displace the binding on the NPFF1 receptor upto 1 uM whereas it displayed a high affinity at the rat spinal cord.Furthermore, several compounds displayed significantly differentaffinities between NPFF1 receptor and the spinal cord membranes. Thesecompounds are highlighted in Table 1 and are ([¹²⁵I]D-Tyr-NPFF,A18Famide, Y8Famide, [Y⁹]A18Famide, Dynorphin A 1-13, Neuropeptide F andMet-Enk-NH2. These data indicate the presence of additional NPFFreceptor subtypes on the rat spinal cord.

[0332] The ability of NPFF1 receptors to functionally couple to PI wastested using intact Cos-7 cells transiently expressing NPFF1. Fulldose-response curves were determined for NPFF-mediated total IP release(FIG. 18A). NPFF stimulated total IP release with an EC50 of 23 nM andan Emax of approximately 200% basal. This weak stimulation was mostprobably mediated by NPFF1 coupling to a Gi/Go G-protein via βγ-inducedPI turnover, since the response was abolished by pretreatment withpertussis toxin but not cholera toxin. In contrast, a robust stimulationof total IP release was observed following NPFF in Cos-7 cellstransfected with both the NPFF1 receptor and the Gq/Gz chimera (FIG.18B). NPFF stimulated total IP release with an EC50 of 2.95 nM, and anEmax of approximately 1500% basal. As anticipated, neither PTX nor CTXattenuated this response. Similar to what was observed in oocytes, thissuggests a coupling in Cos-7 cells to G-proteins of theGα_(i)/Gα_(o)/Gα_(z) class. TABLE 1 pKi for cloned rat NPFF1 receptorbinding in COS-7 cells COMPOUND MEAN SEM n NPFF(F-8-Fa) 8.535 0.02 2(D—Tyr¹—(NMe)Phe³)NPFF 8.549 0.13 4 A18Fa 7.495 0.11 2 PQRFa 8.182 0.032 FMRFa 8.481 0.05 2 YFa 8.382 0.22 2 [Y⁹]A18Fa 7.558 0.12 2 hPP 5.0000.00 2 fPP 5.500 0.35 2 substance P 5.000 0.00 2 Dynorphin A1-13 6.8380.29 2 (3D)Y8Fa 8.623 0.44 4 (2D)Y8Fa 8.330 0.15 4 CCK8 5.000 0.00 2galanin 5.000 0.00 2 dopamine 5.000 0.00 2 naloxone 5.000 0.00 2 CGRP5.000 0.00 2 AF-1 6.634 0.13 2 AF-2 7.023 0.41 2 SchistFLRF 5.960 0.68 2Met5—Arg6—Phe7—Enk—NH2 7.350 0.22 4 Met5—Arg6—Phe7—Enk—OH 5.000 0.00 2Neuropeptide F 6.110 0.06 4 desamino-nor-Y8Ra 7.270 0.10 3 (2DME)Y8Fa9.200 0.01 3 L-arginine 5.000 0.00 1 D-arginine 5.000 0.00 1 desipramine5.000 0.00 1 fenfluramine 5.000 0.00 1 harmine 5.000 0.00 1levocabastine 5.000 0.00 1 ibogaine 5.000 0.00 1 ritanserine 5.000 0.001 a-MSH 5.000 0.00 1 Tyr-MIF-1 5.000 0.00 1 nociceptin 5.000 0.00 1nocistatin 5.000 0.00 1 PMRFa 8.550 0.06 2 FTRF 7.870 0.10 2 FFRF 8.000 0.001 2

[0333] TABLE 2 pKi for rat spinal cord membrane receptor bindingCOMPOUND MEAN SEM n NPFF(F-8-fa) 9.055 0.08 2 (D—Tyr¹—(NMe)Phe³)NPFF*9.724  0.25 4 A18Fa *9.000  0.21 2 PQRFa 8.541 0.07 2 FMRFa 8.493 0.232 Y8Fa *9.189  0.06 2 [Y⁹]A18Fa *8.502  0.01 2 hPP 5.000 0.00 3 fPP*9.118  0.06 3 substance P 5.000 0.00 1 Dynorphin A1-13 *5.700  0.50 2(3D)Y8Fa 9.123 0.12 4 (2D)Y8Fa *9.212  0.23 4 CCK8 5.000 0.00 2 galanin5.000 0.00 2 dopamine 5.000 0.00 2 naloxone 5.000 0.00 2 CGRP 5.000 0.002 AF-1 *7.563  0.47 2 AF-2 *7.965  0.24 2 SchistFLRF 6.390 0.23 2Met—Enk—NH2 *8.400  0.08 4 Met—Enk—OH 5.000 0.00 2 Neuropeptide F*8.100  0.10 3 desamino-nor-Y8Ra 7.51  0.07 3 (2DME) Y8Fa 9.570 0.30 4L-arginine 5.000 0.00 1 D-arginine 5.000 0.00 1 desipiramine 5.000 0.001 fenfluramine 5.000 0.00 1 harmine 5.000 0.00 1 levocabastine 5.0000.00 1 ibogaine 5.000 0.00 1 ritanserine 5.000 0.00 1 a-MSH 5.000 0.00 1Tyr-MIF-1 5.000 0.00 1 nociceptin 5.000 0.00 1 nocistatin 5.000 0.00 1PMRFa 9.370 0.11 2 FTRF 8.160 0.16 2 FFRF 8.980  0.001 2

[0334] Localization

[0335] Detection of mRNA Coding for Rat NPFF1 Receptors:

[0336] mRNA was isolated from multiple tissues (Table 3) and assayed asdescribed. The distribution of mRNA encoding rat NPFF1 receptors iswidespread throughout the central nervous system, and structuresassociated with the nervous system (Table 3, FIGS. 19, 20). The highestlevels of rNPFF1 mRNA are found in the hypothalamus and the pituitarygland. The protected segment seen with mRNA isolated from the pituitary,adrenal gland and ovary is considerably shorter than that seen in othertissue (FIG. 20) and indicates the possibility of splice variants ofthis receptor. Peripheral organs contain little or no mRNA encodingrNPFF1 with the exception of the testes, ovary, the adrenal medulla andthe adrenal cortex. There is good correlation between the distributiondetermined by RT-PCR and RPA (Table 3, FIGS. 19, 20). RT-PCR detectedrat NPFF1 in more areas than RPA as it is a more sensitive technique.

[0337] High levels of mRNA encoding NPFF receptors in the hypothalamusand pituitary, with relatively low expression in most of the otherregions assayed implicates this receptor in neuroendocrine control, aswell as the control of feeding and metabolic regulation. Its presence inother areas, including the spinal cord, medulla and dorsal root gangliaimplicate NPFF receptors as a potential modulator of pain and/or sensorytransmission. Low levels in the hippocampal formation indicate apossible role in learning and memory. TABLE 3 Summary of distribution ofmRNA coding for rat NPFF1 receptors Ribonuclease protection PotentialTissue RT-PCR assay (RPA) applications adrenal + + regulation of cortexsteroid hormones adrenal + ++ regulation of medulla epinephrine releaseurinary − − urinary bladder incontinence duodenum +/− − gastrointestinaldisorders heart +/− − cardiovascular indications kidney + − electrolytebalance, hypertension liver +/− − diabetes lung +/− − respiratorydisorders, asthma ovary + + reproductive function pancreas +/− NAdiabetes, endocrine disorders spleen +/− − immune disorders stomach +/−− gastrointestinal disorders striated +/− − musculoskeletal muscledisorders testicle +/− + reproductive function uterus +/− − reproductivefunction vas deferens − − reproductive function whole brain +++ spinalcord ++ ++ analgesia, sensory modulation and transmission amygdala ++++/− caudate/ ++ − modulation of putamen dopaminergic function cerebellum+++ + motor coordination cerebral ++ + Sensory and motor cortexintegration, cognition DRG + + sensory transmission hippocampus +++ +cognition/memory hypothalamus +++ +++ appetite/obesity, neuroendocrineregulation medulla ++ ++ analgesia, motor coordination olfactory ++ NAolfaction bulb pituitary +++ +++ Endocrine/neuro- endocrine regulationsubstantia +++ ++ Modulation of nigra dopaminergic function superior + −modulation of cervical sympathetic ganglion innervation

[0338] Localization of mRNA Coding for hNPFF2 Receptors Using RT-PCR

[0339] Detection of mRNA Coding for hNPFF2 Receptors

[0340] mRNA was isolated from multiple tissues (Table 4) and assayed asdescribed. The distribution of mRNA encoding hNPFF2 receptors iswidespread throughout all regions assayed. (Table 4, FIG. 21). TABLE 4Distribution of mRNA coding for hNPFF2 receptors Region hNPFF2 PotentialImplications liver ++ Diabetes kidney ++ Hypertension, electrolytebalance Lung ++ Respiratory disorders, asthma heart ++ Cardiovascularindications stomach ++ Gastrointestinal disorders small intestine ++Gastrointestinal disorders spleen ++ Immune function pancreas ++Diabetes, endocrine disorders striated muscle ++ Musculoskeletaldisorders pituitary ++ Endocrine/neuroendocrine regulation whole brain++ amygdala ++ Depression, anxiety, mood disorders hippocampus ++Cognition/memory spinal cord ++ Analgesia, sensory modulation andtransmission cerebellum ++ Motor coordination thalamus ++ sensoryintegration substantia nigra ++ Modulation of dopaminergic function andmotor coordination caudate ++ Modulation of dopaminergic function fetalbrain ++ Developmental disorders fetal lung ++ Developmental disordersfetal kidney ++ Developmental disorders fetal liver ++ Developmentaldisorders HEK-293 cells + HeLa cells − Jurkat cells −

[0341] The cloning of the gene encoding NPFF receptors has provided themeans to explore their physiological roles by pharmacologicalcharacterization, and by Northern and in situ mapping of its mRNAdistribution. Further, the availability of the DNA encoding the NPFFreceptors will facilitate the development of antibodies and antisensetechnologies useful in defining the functions of the gene products invivo. Antisense oligonucleotides which target mRNA molecules toselectively block translation of the gene products in vivo have beenused successfully to relate the expression of a single gene with itsfunctional sequelae. Thus, the cloning of these receptor genes providesthe means to explore their physiological roles in the nervous system andelsewhere, and may thereby help to elucidate structure/functionrelationships within the GPCR superfamily.

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1 42 1 1410 DNA Rattus norvegicus 1 acccttcctg ggccccagtc tacccgcttgaaggtgcccg cctcctttgg agagtgtccc 60 ggagcagaca gtatggaggc ggagccctcccagcctccca acggcagctg gcccctgggt 120 cagaacggga gtgatgtgga gaccagcatggcaaccagcc tcaccttctc ctcctactac 180 caacactcct ctccggtggc agccatgttcatcgcggcct acgtgctcat cttcctcctc 240 tgcatggtgg gcaacaccct ggtctgcttcattgtgctca agaaccggca catgcgcact 300 gtcaccaaca tgtttatcct caacctggccgtcagcgacc tgctggtggg catcttctgc 360 atgcccacaa cccttgtgga caaccttatcactggttggc cttttgacaa cgccacatgc 420 aagatgagcg gcttggtgca gggcatgtccgtgtctgcat cggttttcac actggtggcc 480 atcgctgtgg aaaggttccg ctgcatcgtgcaccctttcc gcgagaagct gacccttcgg 540 aaggcgctgt tcaccatcgc ggtgatctgggctctggcgc tgctcatcat gtgtccctcg 600 gcggtcactc tgacagtcac ccgagaggagcatcacttca tgctggatgc tcgtaaccgc 660 tcctacccgc tctactcgtg ctgggaggcctggcccgaga agggcatgcg caaggtctac 720 accgcggtgc tcttcgcgca catctacctggtgccgctgg cgctcatcgt agtgatgtac 780 gtgcgcatcg cgcgcaagct atgccaggcccccggtcctg cgcgcgacac ggaggaggcg 840 gtggccgagg gtggccgcac ttcgcgccgtagggcccgcg tggtgcacat gctggtcatg 900 gtggcgctct tcttcacgtt gtcctggctgccactctggg tgctgctgct gctcatcgac 960 tatggggagc tgagcgagct gcaactgcacctgctgtcgg tctacgcctt ccccttggca 1020 cactggctgg ccttcttcca cagcagcgccaaccccatca tctacggcta cttcaacgag 1080 aacttccgcc gcggcttcca ggctgccttccgtgcacagc tctgctggcc tccctgggcc 1140 gcccacaagc aagcctactc ggagcggcccaaccgcctcc tgcgcaggcg ggtggtggtg 1200 gacgtgcaac ccagcgactc cggcctgccatcagagtctg gccccagcag cggggtccca 1260 gggcctggcc ggctgccact gcgcaatgggcgtgtggccc atcaggatgg cccgggggaa 1320 gggccaggct gcaaccacat gcccctcaccatcccggcct ggaacatttg aggtggtcca 1380 gagaagggag ggccagtagt cctgtggccc1410 2 432 PRT Rattus norvegicus 2 Met Glu Ala Glu Pro Ser Gln Pro ProAsn Gly Ser Trp Pro Leu Gly 1 5 10 15 Gln Asn Gly Ser Asp Val Glu ThrSer Met Ala Thr Ser Leu Thr Phe 20 25 30 Ser Ser Tyr Tyr Gln His Ser SerPro Val Ala Ala Met Phe Ile Ala 35 40 45 Ala Tyr Val Leu Ile Phe Leu LeuCys Met Val Gly Asn Thr Leu Val 50 55 60 Cys Phe Ile Val Leu Lys Asn ArgHis Met Arg Thr Val Thr Asn Met 65 70 75 80 Phe Ile Leu Asn Leu Ala ValSer Asp Leu Leu Val Gly Ile Phe Cys 85 90 95 Met Pro Thr Thr Leu Val AspAsn Leu Ile Thr Gly Trp Pro Phe Asp 100 105 110 Asn Ala Thr Cys Lys MetSer Gly Leu Val Gln Gly Met Ser Val Ser 115 120 125 Ala Ser Val Phe ThrLeu Val Ala Ile Ala Val Glu Arg Phe Arg Cys 130 135 140 Ile Val His ProPhe Arg Glu Lys Leu Thr Leu Arg Lys Ala Leu Phe 145 150 155 160 Thr IleAla Val Ile Trp Ala Leu Ala Leu Leu Ile Met Cys Pro Ser 165 170 175 AlaVal Thr Leu Thr Val Thr Arg Glu Glu His His Phe Met Leu Asp 180 185 190Ala Arg Asn Arg Ser Tyr Pro Leu Tyr Ser Cys Trp Glu Ala Trp Pro 195 200205 Glu Lys Gly Met Arg Lys Val Tyr Thr Ala Val Leu Phe Ala His Ile 210215 220 Tyr Leu Val Pro Leu Ala Leu Ile Val Val Met Tyr Val Arg Ile Ala225 230 235 240 Arg Lys Leu Cys Gln Ala Pro Gly Pro Ala Arg Asp Thr GluGlu Ala 245 250 255 Val Ala Glu Gly Gly Arg Thr Ser Arg Arg Arg Ala ArgVal Val His 260 265 270 Met Leu Val Met Val Ala Leu Phe Phe Thr Leu SerTrp Leu Pro Leu 275 280 285 Trp Val Leu Leu Leu Leu Ile Asp Tyr Gly GluLeu Ser Glu Leu Gln 290 295 300 Leu His Leu Leu Ser Val Tyr Ala Phe ProLeu Ala His Trp Leu Ala 305 310 315 320 Phe Phe His Ser Ser Ala Asn ProIle Ile Tyr Gly Tyr Phe Asn Glu 325 330 335 Asn Phe Arg Arg Gly Phe GlnAla Ala Phe Arg Ala Gln Leu Cys Trp 340 345 350 Pro Pro Trp Ala Ala HisLys Gln Ala Tyr Ser Glu Arg Pro Asn Arg 355 360 365 Leu Leu Arg Arg ArgVal Val Val Asp Val Gln Pro Ser Asp Ser Gly 370 375 380 Leu Pro Ser GluSer Gly Pro Ser Ser Gly Val Pro Gly Pro Gly Arg 385 390 395 400 Leu ProLeu Arg Asn Gly Arg Val Ala His Gln Asp Gly Pro Gly Glu 405 410 415 GlyPro Gly Cys Asn His Met Pro Leu Thr Ile Pro Ala Trp Asn Ile 420 425 4303 200 DNA Homo sapiens 3 gagccctccc agcctcccaa cagcagttgg cccctaagtcagaatgggac taacactgag 60 gccaccccgg ctacaaacct caccttctcc tcctactatcagcacacctc ccctgtggcg 120 gccatgttca ttgtggccta tgcgctcatc ttcctgctctgcatggtggg caacaccctg 180 gtctgtttca tcgtgctcaa 200 4 66 PRT Homosapiens 4 Glu Pro Ser Gln Pro Pro Asn Ser Ser Trp Pro Leu Ser Gln AsnGly 1 5 10 15 Thr Asn Thr Glu Ala Thr Pro Ala Thr Asn Leu Thr Phe SerSer Tyr 20 25 30 Tyr Gln His Thr Ser Pro Val Ala Ala Met Phe Ile Val AlaTyr Ala 35 40 45 Leu Ile Phe Leu Leu Cys Met Val Gly Asn Thr Leu Val CysPhe Ile 50 55 60 Val Leu 65 5 1302 DNA Homo sapiens 5 gccgacagggctcgccggga gaggttcatc atgaatgaga aatgggacac aaactcttca 60 gaaaactggcatcccatctg gaatgtcaat gacacaaagc atcatctgta ctcagatatt 120 aatattacctatgtgaacta ctatcttcac cagcctcaag tggcagcaat cttcattatt 180 tcctactttctgatcttctt tttgtgcatg atgggaaata ctgtggtttg ctttattgta 240 atgaggaacaaacatatgca cacagtcact aatctcttca tcttaaacct ggccataagt 300 gatttactagttggcatatt ctgcatgcct ataacactgc tggacaatat tatagcagga 360 tggccatttggaaacacgat gtgcaagatc agtggattgg tccagggaat atctgtcgca 420 gcttcagtctttacgttagt tgcaattgct gtagataggt tccagtgtgt ggtctaccct 480 tttaaaccaaagctcactat caagacagcg tttgtcatta ttatgatcat ctgggtccta 540 gccatcaccattatgtctcc atctgcagta atgttacatg tgcaagaaga aaaatattac 600 cgagtgagactcaactccca gaataaaacc agtccagtct actggtgccg ggaagactgg 660 ccaaatcaggaaatgaggaa gatctacacc actgtgctgt ttgccaacat ctacctggct 720 cccctctccctcattgtcat catgtatgga aggattggaa tttcactctt cagggctgca 780 gttcctcacacaggcaggaa gaaccaggag cagtggcacg tggtgtccag gaagaagcag 840 aagatcattaagatgctcct gattgtggcc ctgcttttta ttctctcatg gctgcccctg 900 tggactctaatgatgctctc agactacgct gacctttctc caaatgaact gcagatcatc 960 aacatctacatctacccttt tgcacactgg ctggcattcg gcaacagcag tgtcaatccc 1020 atcatttatggtttcttcaa cgagaatttc cgccgtggtt tccaagaagc tttccagctc 1080 cagctctgccaaaaaagagc aaagcctatg gaagcttatg ccctaaaagc taaaagccat 1140 gtgctcataaacacatctaa tcagcttgtc caggaatcta catttcaaaa ccctcatggg 1200 gaaaccttgctttataggaa aagtgctgaa aaaccccaac aggaattagt gatggaagaa 1260 ttaaaagaaactactaacag cagtgagatt taaaaagagc ta 1302 6 420 PRT Homo sapiens 6 MetAsn Glu Lys Trp Asp Thr Asn Ser Ser Glu Asn Trp His Pro Ile 1 5 10 15Trp Asn Val Asn Asp Thr Lys His His Leu Tyr Ser Asp Ile Asn Ile 20 25 30Thr Tyr Val Asn Tyr Tyr Leu His Gln Pro Gln Val Ala Ala Ile Phe 35 40 45Ile Ile Ser Tyr Phe Leu Ile Phe Phe Leu Cys Met Met Gly Asn Thr 50 55 60Val Val Cys Phe Ile Val Met Arg Asn Lys His Met His Thr Val Thr 65 70 7580 Asn Leu Phe Ile Leu Asn Leu Ala Ile Ser Asp Leu Leu Val Gly Ile 85 9095 Phe Cys Met Pro Ile Thr Leu Leu Asp Asn Ile Ile Ala Gly Trp Pro 100105 110 Phe Gly Asn Thr Met Cys Lys Ile Ser Gly Leu Val Gln Gly Ile Ser115 120 125 Val Ala Ala Ser Val Phe Thr Leu Val Ala Ile Ala Val Asp ArgPhe 130 135 140 Gln Cys Val Val Tyr Pro Phe Lys Pro Lys Leu Thr Ile LysThr Ala 145 150 155 160 Phe Val Ile Ile Met Ile Ile Trp Val Leu Ala IleThr Ile Met Ser 165 170 175 Pro Ser Ala Val Met Leu His Val Gln Glu GluLys Tyr Tyr Arg Val 180 185 190 Arg Leu Asn Ser Gln Asn Lys Thr Ser ProVal Tyr Trp Cys Arg Glu 195 200 205 Asp Trp Pro Asn Gln Glu Met Arg LysIle Tyr Thr Thr Val Leu Phe 210 215 220 Ala Asn Ile Tyr Leu Ala Pro LeuSer Leu Ile Val Ile Met Tyr Gly 225 230 235 240 Arg Ile Gly Ile Ser LeuPhe Arg Ala Ala Val Pro His Thr Gly Arg 245 250 255 Lys Asn Gln Glu GlnTrp His Val Val Ser Arg Lys Lys Gln Lys Ile 260 265 270 Ile Lys Met LeuLeu Ile Val Ala Leu Leu Phe Ile Leu Ser Trp Leu 275 280 285 Pro Leu TrpThr Leu Met Met Leu Ser Asp Tyr Ala Asp Leu Ser Pro 290 295 300 Asn GluLeu Gln Ile Ile Asn Ile Tyr Ile Tyr Pro Phe Ala His Trp 305 310 315 320Leu Ala Phe Gly Asn Ser Ser Val Asn Pro Ile Ile Tyr Gly Phe Phe 325 330335 Asn Glu Asn Phe Arg Arg Gly Phe Gln Glu Ala Phe Gln Leu Gln Leu 340345 350 Cys Gln Lys Arg Ala Lys Pro Met Glu Ala Tyr Ala Leu Lys Ala Lys355 360 365 Ser His Val Leu Ile Asn Thr Ser Asn Gln Leu Val Gln Glu SerThr 370 375 380 Phe Gln Asn Pro His Gly Glu Thr Leu Leu Tyr Arg Lys SerAla Glu 385 390 395 400 Lys Pro Gln Gln Glu Leu Val Met Glu Glu Leu LysGlu Thr Thr Asn 405 410 415 Ser Ser Glu Ile 420 7 1293 DNA Homo sapiens7 atggaggggg agccctccca gcctcccaac agcagttggc ccctaagtca gaatgggact 60aacactgagg ccaccccggc tacaaacctc accttctcct cctactatca gcacacctcc 120cctgtggcgg ccatgttcat tgtggcctat gcgctcatct tcctgctctg catggtgggc 180aacaccctgg tctgtttcat cgtgctcaag aaccggcaca tgcatactgt caccaacatg 240ttcatcctca acctggctgt cagtgacctg ctggtgggca tcttctgcat gcccaccacc 300cttgtggaca acctcatcac tgggtggccc ttcgacaatg ccacatgcaa gatgagcggc 360ttggtgcagg gcatgtctgt gtcggcttcc gttttcacac tggtggccat tgctgtggaa 420aggttccgct gcatcgtgca ccctttccgc gagaagctga ccctgcggaa ggcgctcgtc 480accatcgccg tcatctgggc cctggcgctg ctcatcatgt gtccctcggc cgtcacgctg 540accgtcaccc gtgaggagca ccacttcatg gtggacgccc gcaaccgctc ctaccctctc 600tactcctgct gggaggcctg gcccgagaag ggcatgcgca gggtctacac cactgtgctc 660ttctcgcaca tctacctggc gccgctggcg ctcatcgtgg tcatgtacgc ccgcatcgcg 720cgcaagctct gccaggcccc gggcccggcc cccgggggcg aggaggctgc ggacccgcga 780gcatcgcggc gcagagcgcg cgtggtgcac atgctggtca tggtggcgct gttcttcacg 840ctgtcctggc tgccgctctg ggcgctgctg ctgctcatcg actacgggca gctcagcgcg 900ccgcagctgc acctggtcac cgtctacgcc ttccccttcg cgcactggct ggccttcttc 960aacagcagcg ccaaccccat catctacggc tacttcaacg agaacttccg ccgcggcttc 1020caggccgcct tccgcgcccg cctctgcccg cgcccgtcgg ggagccacaa ggaggcctac 1080tccgagcggc ccggcgggct tctgcacagg cgggtcttcg tggtggtgcg gcccagcgac 1140tccgggctgc cctctgagtc gggccctagc agtggggccc ccaggcccgg ccgcctcccg 1200ctgcggaatg ggcgggtggc tcaccacggc ttgcccaggg aagggcctgg ctgctcccac 1260ctgcccctca ccattccagc ctgggatatc tga 1293 8 430 PRT Homo sapiens 8 MetGlu Gly Glu Pro Ser Gln Pro Pro Asn Ser Ser Trp Pro Leu Ser 1 5 10 15Gln Asn Gly Thr Asn Thr Glu Ala Thr Pro Ala Thr Asn Leu Thr Phe 20 25 30Ser Ser Tyr Tyr Gln His Thr Ser Pro Val Ala Ala Met Phe Ile Val 35 40 45Ala Tyr Ala Leu Ile Phe Leu Leu Cys Met Val Gly Asn Thr Leu Val 50 55 60Cys Phe Ile Val Leu Lys Asn Arg His Met His Thr Val Thr Asn Met 65 70 7580 Phe Ile Leu Asn Leu Ala Val Ser Asp Leu Leu Val Gly Ile Phe Cys 85 9095 Met Pro Thr Thr Leu Val Asp Asn Leu Ile Thr Gly Trp Pro Phe Asp 100105 110 Asn Ala Thr Cys Lys Met Ser Gly Leu Val Gln Gly Met Ser Val Ser115 120 125 Ala Ser Val Phe Thr Leu Val Ala Ile Ala Val Glu Arg Phe ArgCys 130 135 140 Ile Val His Pro Phe Arg Glu Lys Leu Thr Leu Arg Lys AlaLeu Val 145 150 155 160 Thr Ile Ala Val Ile Trp Ala Leu Ala Leu Leu IleMet Cys Pro Ser 165 170 175 Ala Val Thr Leu Thr Val Thr Arg Glu Glu HisHis Phe Met Val Asp 180 185 190 Ala Arg Asn Arg Ser Tyr Pro Leu Tyr SerCys Trp Glu Ala Trp Pro 195 200 205 Glu Lys Gly Met Arg Arg Val Tyr ThrThr Val Leu Phe Ser His Ile 210 215 220 Tyr Leu Ala Pro Leu Ala Leu IleVal Val Met Tyr Ala Arg Ile Ala 225 230 235 240 Arg Lys Leu Cys Gln AlaPro Gly Pro Ala Pro Gly Gly Glu Glu Ala 245 250 255 Ala Asp Pro Arg AlaSer Arg Arg Arg Ala Arg Val Val His Met Leu 260 265 270 Val Met Val AlaLeu Phe Phe Thr Leu Ser Trp Leu Pro Leu Trp Ala 275 280 285 Leu Leu LeuLeu Ile Asp Tyr Gly Gln Leu Ser Ala Pro Gln Leu His 290 295 300 Leu ValThr Val Tyr Ala Phe Pro Phe Ala His Trp Leu Ala Phe Phe 305 310 315 320Asn Ser Ser Ala Asn Pro Ile Ile Tyr Gly Tyr Phe Asn Glu Asn Phe 325 330335 Arg Arg Gly Phe Gln Ala Ala Phe Arg Ala Arg Leu Cys Pro Arg Pro 340345 350 Ser Gly Ser His Lys Glu Ala Tyr Ser Glu Arg Pro Gly Gly Leu Leu355 360 365 His Arg Arg Val Phe Val Val Val Arg Pro Ser Asp Ser Gly LeuPro 370 375 380 Ser Glu Ser Gly Pro Ser Ser Gly Ala Pro Arg Pro Gly ArgLeu Pro 385 390 395 400 Leu Arg Asn Gly Arg Val Ala His His Gly Leu ProArg Glu Gly Pro 405 410 415 Gly Cys Ser His Leu Pro Leu Thr Ile Pro AlaTrp Asp Ile 420 425 430 9 23 DNA Artificial Sequence Description ofArtificial Sequence primer/probe 9 gyntwyrynn tnwsntgght ncc 23 10 23DNA Artificial Sequence Description of Artificial Sequence primer/probe10 avnadngbrw avannanngg rtt 23 11 25 DNA Artificial SequenceDescription of Artificial Sequence primer/probe 11 ttatgcttcc ggctcgtatgttgtg 25 12 26 DNA Artificial Sequence Description of ArtificialSequence primer/probe 12 atgtgctgca aggcgattaa gttggg 26 13 26 DNAArtificial Sequence Description of Artificial Sequence primer/probe 13ggtgctgctg ctgctcatcg actatg 26 14 26 DNA Artificial SequenceDescription of Artificial Sequence primer/probe 14 ttggcgctgc tgtggaagaaggccag 26 15 24 DNA Artificial Sequence Description of ArtificialSequence primer/probe 15 cggtgctctt cgcgcacatc tacc 24 16 60 DNAArtificial Sequence Description of Artificial Sequence primer/probe 16tgccaagggg aaggcgtaga ccgacagcag gtgcagttgc agctcgatca gctccccata 60 1753 DNA Artificial Sequence Description of Artificial Sequenceprimer/probe 17 ccacccttgt ggacaacctc atcactgggt ggcccttcga caatgccacatgc 53 18 24 DNA Artificial Sequence Description of Artificial Sequenceprimer/probe 18 ctgctctgca tggtgggcaa cacc 24 19 21 DNA ArtificialSequence Description of Artificial Sequence primer/probe 19 gacggcgatggtgacgagcg c 21 20 65 DNA Artificial Sequence Description of ArtificialSequence primer/probe 20 gtcaccaaca tgttcatcct caacctggct gtcagtgacctgctggtggg catcttctgc 60 atgcc 65 21 24 DNA Artificial SequenceDescription of Artificial Sequence primer/probe 21 gcgagaagct gaccctgcggaagg 24 22 24 DNA Artificial Sequence Description of Artificial Sequenceprimer/probe 22 tcgtcaccat cgccgtcatc tggg 24 23 24 DNA ArtificialSequence Description of Artificial Sequence primer/probe 23 cgtcatctgggccgagggac acag 24 24 23 DNA Artificial Sequence Description ofArtificial Sequence primer/probe 24 tgacggcgat ggtgacgagc gcc 23 25 23DNA Artificial Sequence Description of Artificial Sequence primer/probe25 cagcctccca acagcagttg gcc 23 26 35 DNA Artificial SequenceDescription of Artificial Sequence primer/probe 26 tagcaaggat ccgcatatggagggggagcc ctccc 35 27 36 DNA Artificial Sequence Description ofArtificial Sequence primer/probe 27 cttcatgaat tcatcgcctg catgtatctcgtgtcc 36 28 31 DNA Artificial Sequence Description of ArtificialSequence primer/probe 28 cgtgtacggt gggaggtcta tataagcaga g 31 29 27 DNAArtificial Sequence Description of Artificial Sequence primer/probe 29ccatcctaat acgactcact atagggc 27 30 23 DNA Artificial SequenceDescription of Artificial Sequence primer/probe 30 actcactata gggctcgagcggc 23 31 26 DNA Artificial Sequence Description of Artificial Sequenceprimer/probe 31 tgatagtgag ctttggttta aaaggg 26 32 26 DNA ArtificialSequence Description of Artificial Sequence primer/probe 32 gaagatctacaccactgtgc tgtttg 26 33 25 DNA Artificial Sequence Description ofArtificial Sequence primer/probe 33 aacatctacc tggctcccct ctccc 25 34 25DNA Artificial Sequence Description of Artificial Sequence primer/probe34 ttgtcatcat gtatggaagg attgg 25 35 24 DNA Artificial SequenceDescription of Artificial Sequence primer/probe 35 gaccacacac tggaacctatctac 24 36 25 DNA Artificial Sequence Description of Artificial Sequenceprimer/probe 36 gcaattgcaa ctaacgtaaa gactg 25 37 37 DNA ArtificialSequence Description of Artificial Sequence primer/probe 37 tagcaaggatccgaggttca tcatgaatga gaaatgg 37 38 36 DNA Artificial SequenceDescription of Artificial Sequence primer/probe 38 cttcatgaat tcgcgtagtagagttaggat tatcac 36 39 24 DNA Artificial Sequence Description ofArtificial Sequence primer/probe 39 ctcctactac caacactcct ctcc 24 40 19DNA Artificial Sequence Description of Artificial Sequence primer/probe40 acgggttacg agcatccag 19 41 27 DNA Artificial Sequence Description ofArtificial Sequence primer/probe 41 gatcagtgga ttggtccagg gaatatc 27 4225 DNA Artificial Sequence Description of Artificial Sequenceprimer/probe 42 ccaggtagat gttggcaaac agcac 25

What is claimed is:
 1. An isolated nucleic acid encoding a mammalianNPFF receptor.
 2. The nucleic acid of claim 1, wherein the nucleic acidis DNA.
 3. The DNA of claim 2, wherein the DNA is cDNA.
 4. The DNA ofclaim 2, wherein the DNA is genomic DNA.
 5. The nucleic acid of claim 1,wherein the nucleic acid is RNA.
 6. The nucleic acid of claim 1, whereinthe mammalian NPFF receptor is a NPFF1 receptor.
 7. The nucleic acid ofclaim 6, wherein the mammalian NPFF1 receptor is a rat NPFF1 receptor.8. The nucleic acid of claim 6, wherein the mammalian NPFF1 receptor isa human NPFF1 receptor.
 9. The nucleic acid of claim 1, wherein themammalian NPFF receptor is a NPFF2 receptor.
 10. The nucleic acid ofclaim 9, wherein the mammalian NPFF2 receptor is a human NPFF2 receptor.11. The nucleic acid of claim 7, wherein the rat NPFF1 receptor has anamino acid sequence identical to that encoded by the plasmid pEXJ-rNPFF1(ATCC Accession No. 203184).
 12. The nucleic acid of claim 7, whereinthe rat NPFF1 receptor has an amino acid sequence identical to the aminoacid sequence shown in FIG. 2 (Seq. I.D. No. 2).
 13. The nucleic acid ofclaim 8, wherein the human NPFF1 receptor has an amino acid sequenceidentical to that encoded by plasmid pWE15-hNPFF1 (ATCC Accession No.203183).
 14. The nucleic acid of claim 8, wherein the human NPFF1receptor has an amino acid sequence identical to the amino acid sequenceshown in FIG. 5 (Seq. I.D. No. 4).
 15. The nucleic acid of claim 8,wherein the human NPFF1 receptor has an amino acid sequence identical tothat encoded by plasmid pcDNA3.1-hNPFF1 (ATCC Accession No. 203605). 16.The nucleic acid of claim 8, wherein the human NPFF1 receptor has anamino acid sequence identical to the amino acid sequence shown in FIG.12 (Seq. I.D. No. 8).
 17. The nucleic acid of claim 10, wherein thehuman NPFF2 receptor has an amino acid sequence identical to thatencoded by plasmid pCDNA3.1-hNPFF2b (ATCC Accession No. 203255).
 18. Thenucleic acid of claim 10, wherein the human NPFF2 receptor has an aminoacid sequence identical to the amino acid sequence shown in FIG. 8 (Seq.I.D. No. 6).
 19. The nucleic acid of claim 1, wherein the nucleic acid(a) hybridizes to a nucleic acid having the defined sequence shown inFIG. 1 (Seq. I.D. No. 1) under low stringency conditions or a sequencecomplementary thereto and (b) is further characterized by its ability tocause a change in the pH of a culture of CHO cells when a NPFF peptideis added to the culture and the CHO cells express the nucleic acid whichhybridized to the nucleic acid having the defined sequence of itscomplement.
 20. The nucleic acid of claim 1, wherein the nucleic acid(a) hybridizes to a nucleic acid having the defined sequence shown inFIG. 4 (Seq. I.D. No. 3) under low stringency conditions or a sequencecomplementary thereto and (b) is further characterized by its ability tocause a change in the pH of a culture of CHO cells when a NPFF peptideis added to the culture and the CHO cells express the nucleic acid whichhybridized to the nucleic acid having the defined sequence or itscomplement.
 21. The nucleic acid of claim 1, wherein the nucleic acid(a) hybridizes to a nucleic acid having the defined sequence shown inFIG. 7 (Seq. ID No. 5) under low stringency conditions or a sequencecomplementary thereto and (b) id further characterized by its ability tocause a change in the pH of a culture of CHO cells when a NPFF peptideis added to the culture and the CHO cells express the nucleic acid whichhybridized to the nucleic acid having the defined sequence or itscomplement.
 22. The nucleic acid of claim 1, wherein the nucleic acid(a) hybridizes to a nucleic acid having the defined sequence shown inFIG. 11 (Seq. I.D. No. 7) under low stringency conditions or a sequencecomplementary thereto and (b) is further characterized by its ability tocause a change in the pH of a culture of CHO cells when a NPFF peptideis added to the culture and the CHO cells express the nucleic acid whichhybridized to the nucleic acid having the defined sequence or itscomplement.
 23. A purified mammalian NPFF receptor protein.
 24. Thepurified mammalian NPFF receptor protein of claim 23, wherein the NPFFreceptor protein is a NPFF1 receptor protein.
 25. The purified mammalianNPFF receptor protein of claim 23, wherein the NPFF receptor protein isa NPFF2 receptor protein.
 26. The purified mammalian NPFF1 receptorprotein of claim 24, wherein the NPFF1 receptor protein is a rat NPFF1receptor protein.
 27. The purified mammalian NPFF1 receptor protein ofclaim 24, wherein the NPFF1 receptor protein is a human NPFF1 receptorprotein.
 28. The purified mammalian NPFF2 receptor protein of claim 25,wherein the NPFF2 receptor protein is a human NPFF2 receptor protein.29. A vector comprising the nucleic acid of claim
 1. 30. A vectorcomprising the nucleic acid of claim
 6. 31. A vector comprising thenucleic acid of claim
 9. 32. A vector comprising the nucleic acid of anyof claims 19, 20, 21, or
 22. 33. A vector of any of claims 19, 20, 21,22, 29, 30, or 31 adapted for expression in a cell which comprises theregulatory elements necessary for expression of the nucleic acid in thecell operatively linked to the nucleic acid encoding the receptor so asto permit expression thereof, wherein the cell is a bacterial,amphibian, yeast, insect or mammalian cell.
 34. The vector of claim 33,wherein the vector is a baculovirus.
 35. The vector of claim 29, whereinthe vector is a plasmid.
 36. The plasmid of claim 35 designatedpEXJ-rNPFF1 (ATCC Accession No. 203184).
 37. The plasmid of claim 35designated pWE15-hNPFF1 (ATCC Accession No. 203183).
 38. The plasmid ofclaim 35 designated pCDNA3.1-hNPFF2b (ATCC Accession No. 203255). 39.The plasmid of claim 35 designated pcDNA3.1-hNPFF1 (ATCC Accession No.203605).
 40. A cell comprising the vector of claim
 33. 41. A cell ofclaim 40, wherein the cell is a non-mammalian cell.
 42. A cell of claim41, wherein the non-mammalian cell is a Xenopus oocyte cell or a Xenopusmelanophore cell.
 43. A cell of claim 40, wherein the cell is amammalian cell.
 44. A mammalian cell of claim 43, wherein the cell is aCOS-7 cell, a 293 human embryonic kidney cell, a NIH-3T3 cell, a LM(tk-)cell, a mouse Y1 cell, or a CHO cell.
 45. An insect cell comprising thevector of claim
 33. 46. An insect cell of claim 45, wherein the insectcell is an Sf9 cell, an Sf21 cell or a Trichoplusia ni 5B1-4 cell.
 47. Amembrane preparation isolated from the cell of any of claims 40, 41, 43,44, 45 or
 46. 48. A nucleic acid probe comprising at least 15nucleotides, which probe specifically hybridizes with a nucleic acidencoding a mammalian NPFF receptor, wherein the probe has a uniquesequence corresponding to a sequence present within one of the twostrands of the nucleic acid encoding the mammalian NPFF1 receptor andcontained in plasmid pEXJ-rNPFF1 (ATCC Accession No. 203184),plasmidpWE15-hNPFF1 (ATCC Accession No. 203183), plasmid pCDNA3.1-hNPFF2b (ATCCAccession No. 203255), or plasmid pcDNA3.1-hNPFF1 (ATCC Accession No.203605).
 49. A nucleic acid probe comprising at least 15 nucleotides,which probe specifically hybridizes with a nucleic acid encoding amammalian NPFF receptor, wherein the probe has a unique sequencecorresponding to a sequence present within (a) the nucleic acid sequenceshown in FIG. 1 (Seq. I.D. No. 1) or (b) the reverse complement thereto.50. A nucleic acid probe comprising at least 15 nucleotides, which probespecifically hybridizes with a nucleic acid encoding a mammalian NPFFreceptor, wherein the probe has a unique sequence corresponding to asequence present within (a) the nucleic acid sequence shown in FIG. 4(Seq. I.D. No. 3) or (b) the reverse complement thereto.
 51. A nucleicacid probe comprising at least 15 nucleotides, which probe specificallyhybridizes with a nucleic acid encoding a mammalian NPFF receptor,wherein the probe has a unique sequence corresponding to a sequencepresent within (a) the nucleic acid sequence shown in FIG. 7 (Seq. I.D.No. 5) or (b) the reverse complement thereto.
 52. A nucleic acid probecomprising at least 15 nucleotides, which probe specifically hybridizeswith a nucleic acid encoding a mammalian NPFF receptor, wherein theprobe has a unique sequence corresponding to a sequence present within(a) the nucleic acid sequence shown in FIG. 11 (Seq. I.D. No. 7) or (b)the reverse complement thereto.
 53. The nucleic acid probe of claim 49,50, 51, or 52, wherein the nucleic acid is DNA.
 54. The nucleic acidprobe of claim 49, 50, 51, or 52, wherein the nucleic acid is RNA. 55.An antisense oligonucleotide having a sequence capable of specificallyhybridizing to the RNA of claim 5, so as to prevent translation of theRNA.
 56. An antisense oligonucleotide having a sequence capable ofspecifically hybridizing to the genomic DNA of claim 4, so as to preventtranscription of the genomic DNA.
 57. An antisense oligonucleotide ofclaim 55 or 56, wherein the oligonucleotide comprises chemicallymodified nucleotides or nucleotide analogues.
 58. An antibody capable ofbinding to a mammalian NPFF receptor encoded by the nucleic acid ofclaim
 1. 59. An antibody of claim 58, wherein the mammalian NPFFreceptor is a human NPFF1 receptor.
 60. An antibody of claim 58, whereinthe mammalian NPFF receptor is a rat NPFF1 receptor.
 61. An antibody ofclaim
 58. wherein the mammalian NPFF receptor is a human NPFF2 receptor.62. An agent capable of competitively inhibiting the binding of theantibody of claim 58 to a mammalian NPFF receptor.
 63. An antibody ofclaim 58, wherein the antibody is a monoclonal antibody or antisera. 64.A pharmaceutical composition comprising (a) an amount of theoligonucleotide of claim 55 capable of passing through a cell membraneand effective to reduce expression of a mammalian NPFF receptor and (b)a pharmaceutically acceptable carrier capable of passing through thecell membrane.
 65. A pharmaceutical composition of claim 64, wherein theoligonucleotide is coupled to a substance which inactivates mRNA.
 66. Apharmaceutical composition of claim 65, wherein the substance whichinactivates mRNA is a ribozyme.
 67. A pharmaceutical composition ofclaim 65, wherein the pharmaceutically acceptable carrier comprises astructure which binds to a mammalian NPFF receptor on a cell capable ofbeing taken up by the cells after binding to the structure.
 68. Apharmaceutical composition of claim 67, wherein the pharmaceuticallyacceptable carrier is capable of binding to a mammalian NPFF receptorwhich is specific for a selected cell type.
 69. A pharmaceuticalcomposition which comprises an amount of the antibody of claim 58effective to block binding of a ligand to a human NPFF receptor and apharmaceutically acceptable carrier.
 70. A transgenic, nonhuman mammalexpressing DNA encoding a mammalian NPFF receptor of claim
 1. 71. Atransgenic, nonhuman mammal comprising a homologous recombinationknockout of the native mammalian NPFF receptor.
 72. A transgenic,nonhuman mammal whose genome comprises antisense DNA complementary tothe DNA encoding a mammalian NPFF receptor of claim 1 so placed withinthe genome as to be transcribed into antisense mRNA which iscomplementary to mRNA encoding the mammalian NPFF receptor and whichhybridizes to mRNA encoding the mammalian NPFF receptor, therebyreducinq its translation.
 73. The transgenic, nonhuman mammal of claim70 or 71, wherein the DNA encoding the mammalian NPFF receptoradditionally comprises an inducible promoter.
 74. The transgenic,nonhuman mammal of claim 70 or 71, wherein the DNA encoding themammalian NPFF receptor additionally comprises tissue specificregulatory elements.
 75. A transgenic, nonhuman mammal of claim 70, 71,or 72, wherein the transgenic, nonhuman mammal is a mouse.
 76. A processfor identifying a chemical compound which specifically binds to amammalian NPFF receptor which comprises contacting cells containing DNAencoding and expressing on their cell surface the mammalian NPFFreceptor, wherein such cells do not normally express the mammalian NPFFreceptor, with the compound under conditions suitable for binding, anddetecting specific binding of the chemical compound to the mammalianNPFF receptor.
 77. A process for identifying a chemical compound whichspecifically binds to a mammalian NPFF receptor which comprisescontacting a membrane preparation from cells containing DNA encoding andexpressing on their cell surface the mammalian NPFF receptor, whereinsuch cells do not normally express the mammalian NPFF receptor, with thecompound under conditions suitable for binding, and detecting specificbinding of the chemical compound to the mammalian NPFF receptor.
 78. Theprocess of claim 76 or 77, wherein the mammalian NPFF receptor is ahuman NPFF1 receptor.
 79. The process of claim 76 or 77, wherein themammalian NPFF receptor is a human NPFF2 receptor.
 80. The process ofclaim 76 or 77, wherein the mammalian NPFF receptor has substantiallythe same amino acid sequence as the human NPFF1 receptor encoded byplasmid pWE15-hNPFF1 (ATCC Accession No. 203183).
 81. The process ofclaim 76 or 77, wherein the mammalian NPFF receptor has substantiallythe same amino acid sequence as the human NPFF1 receptor encoded byplasmid pcDNA3.1-hNPFF1 (ATCC Accession No. 203605).
 82. The process ofclaim 65 or 66, wherein the mammalian NPFF receptor has substantiallythe same amino acid sequence as the human NPFF2 receptor encoded byplasmid pCDNA3.1-hNPFF2b (ATCC Accession No. 203255).
 83. The process ofclaim 76 or 77, wherein the mammalian NPFF receptor has substantiallythe same amino acid sequence as that shown in FIG. 5 (Seq. I.D. No. 4).84. The process of claim 76 or 77, wherein the mammalian NPFF receptorhas the amino acid sequence shown in FIG. 5 (Seq. I.D. No. 4).
 85. Theprocess of claim 76 or 77, wherein the mammalian NPFF receptor hassubstantially the same amino acid sequence as that shown in FIG. 8 (Seq.I.D. No. 6).
 86. The process of claim 76 or 77, wherein the mammalianNPFF receptor has the same amino acid sequence shown in FIG. 8 (Seq.I.D. No. 6).
 87. The process of claim 76 or 77, wherein the mammalianNPFF receptor has substantially the same amino acid sequence as thatshown in FIG. 12 (Seq. I.D. No. 8).
 88. The process of claim 76 or 77,wherein the mammalian NPFF receptor has the same amino acid sequenceshown in FIG. 12 (Seq. I.D. No. 8).
 89. The process of claim 76 or 77,wherein the compound is not previously known to bind to a mammalian NPFFreceptor.
 90. A compound identified by the process of claim
 89. 91. Aprocess of claim 76 or 77, wherein the cell is an insect cell.
 92. Theprocess of claim 76 or 77, wherein the cell is a mammalian cell.
 93. Theprocess of claim 92, wherein the cell is nonneuronal in origin.
 94. Theprocess of claim 93, wherein the nonneuronal cell is a COS-7 cell, 293human embryonic kidney cell, a CHO cell, a NIH-3T3 cell, a mouse Y1cell, or a LM(tk-) cell.
 95. A process of claim 92, wherein the compoundis a compound not previously known to bind to a mammalian NPFF receptor.96. A compound identified by the process of claim
 95. 97. A processinvolving competitive binding for identifying a chemical compound whichspecifically binds to a mammalian NPFF receptor which comprisesseparately contacting cells expressing on their cell surface themammalian NPFF receptor, wherein such cells do not normally express themammalian NPFF receptor, with both the chemical compound and a secondchemical compound known to bind to the receptor, and with only thesecond chemical compound, under conditions suitable for binding of bothcompounds, and detecting specific binding of the chemical compound tothe mammalian NPFF receptor, a decrease in the binding of the secondchemical compound to the mammalian NPFF receptor in the presence of thechemical compound indicating that the chemical compound binds to themammalian NPFF receptor.
 98. A process involving competitive binding foridentifying a chemical compound which specifically binds to a mammalianNPFF receptor which comprises separately contacting a membranepreparation from cells expressing on their cell surface the mammalianNPFF receptor, wherein such cells do not normally express the mammalianNPFF receptor, with both the chemical compound and a second chemicalcompound known to bind to the receptor, and with only the secondchemical compound, under conditions suitable for binding of bothcompounds, and detecting specific binding of the chemical compound tothe mammalian NPFF receptor, a decrease in the binding of the secondchemical compound to the mammalian NPFF receptor in the presence of thechemical compound indicating that the chemical compound binds to themammalian NPFF receptor.
 99. A process of claim 97 or 98, wherein themammalian NPFF receptor is a human NPFF1 receptor.
 100. A process ofclaim 97 or 98, wherein the mammalian NPFF receptor is a human NPFF2receptor.
 101. The process of claim 97 or 98, wherein the cell is aninsect cell.
 102. The process of claim 97 or 98, wherein the cell is amammalian cell.
 103. The process of claim 102, wherein the cell isnonneuronal in origin.
 104. The process of claim 103, wherein thenonneuronal cell is a COS-7 cell, 293 human embryonic kidney cell, a CHOcell, a NIH-3T3 cell, a mouse Y1 cell, or a LM(tk-) cell.
 105. Theprocess of claim 104, wherein the compound is not previously known tobind to a mammalian NPFF receptor.
 106. A compound identified by theprocess of claim
 105. 107. A method of screening a plurality of chemicalcompounds not known to bind to a mammalian NPFF receptor to identify acompound which specifically binds to the mammalian NPFF receptor, whichcomprises (a) contacting cells transfected with and expressing DNAencoding the mammalian NPFF receptor with a compound known to bindspecifically to the mammalian NPFF receptor; (b) contacting thepreparation of step (a) with the plurality of compounds not known tobind specifically to the mammalian NPFF receptor, under conditionspermitting binding of compounds known to bind to the mammalian NPFFreceptor; (c) determining whether the binding of the compound known tobind to the mammalian NPFF receptor is reduced in the presence of anycompound within the plurality of compounds, relative to the binding ofthe compound in the absence of the plurality of compounds; and if so (d)separately determining the binding to the mammalian NPFF receptor ofcompounds included in the plurality of compounds, so as to therebyidentify the compound which specifically binds to the mammalian NPFFreceptor.
 108. A method of screening a plurality of chemical compoundsnot known to bind to a mammalian NPFF receptor to identify a compoundwhich specifically binds to the mammalian NPFF receptor, which comprises(a) contacting a membrane preparation from cells transfected with andexpressing DNA encoding the mammalian NPFF receptor with the pluralityof compounds not known to bind specifically to the mammalian NPFFreceptor under conditions permitting binding of compounds known to bindto the mammalian NPFF receptor; (b) determining whether the binding of acompound known to bind to the mammalian NPFF receptor is reduced in thepresence of any compound within the plurality of compounds, relative tothe binding of the compound in the absence of the plurality ofcompounds; and if so (c) separately determining the binding to themammalian NPFF receptor of compounds included in the plurality ofcompounds, so as to thereby identify the compound which specificallybinds to the mammalian NPFF receptor.
 109. A method of claim 107 or 108,wherein the mammalian NPFF receptor is a human NPFF1 receptor.
 110. Amethod of claim 107 or 108, wherein the mammalian NPFF receptor is ahuman NPFF2 receptor.
 111. A method of claim 107, 108, 109, or 110,wherein the cell is a mammalian cell.
 112. A method of claim 111,wherein the mammalian cell is non-neuronal in origin.
 113. The method ofclaim 112, wherein the non-neuronal cell is a COS-7 cell, a 293 humanembryonic kidney cell, a LM(tk-) cell, a CHO cell, a mouse Y1 cell, oran NIH-3T3 cell.
 114. A method of detecting expression of a mammalianNPFF receptor by detecting the presence of mRNA coding for the mammalianNPFF receptor which comprises obtaining total mRNA from the cell andcontacting the mRNA so obtained with the nucleic acid probe of any ofclaims 48, 49, 50, 51, or 52 under hybridizing conditions, detecting thepresence of mRNA hybridizing to the probe, and thereby detecting theexpression of the mammalian NPFF receptor by the cell.
 115. A method ofdetecting the presence of a mammalian NPFF receptor on the surface of acell which comprises contacting the cell with the antibody of claim 58under conditions permitting binding of the antibody to the receptor,detecting the presence of the antibody bound to the cell, and therebydetecting the presence of the mammalian NPFF receptor on the surface ofthe cell.
 116. A method of determining the physiological effects ofvarying levels of activity of mammalian NPFF receptors which comprisesproducing a transgenic, nonhuman mammal of claim 73 whose levels ofmammalian NPFF receptor activity are varied by use of an induciblepromoter which regulates mammalian NPFF receptor expression.
 117. Amethod of determining the physiological effects of varying levels ofactivity of mammalian NPFF receptors which comprises producing a panelof transgenic, nonhuman mammals of claim 73 each expressing a differentamount of mammalian NPFF receptor.
 118. A method for identifying anantagonist capable of alleviating an abnormality wherein the abnormalityis alleviated by decreasing the activity of a mammalian NPFF receptorcomprising administering a compound to the transgenic, nonhuman mammalof claim 70, 73, 74, or 75, and determining whether the compoundalleviates the physical and behavioral abnormalities displayed by thetransgenic, nonhuman mammal as a result of overactivity of a mammalianNPFF receptor, the alleviation of the abnormality identifying thecompound as an antagonist.
 119. The method of claim 118, wherein themammalian NPFF receptor is a human NPFF1 receptor.
 120. The method ofclaim 118, wherein the mammalian NPFF receptor is a human NPFF2receptor.
 121. An antagonist identified by the method of claim
 118. 122.A pharmaceutical composition comprising an antagonist of claim 121 and apharmaceutically acceptable carrier.
 123. A method of treating anabnormality in a subject wherein the abnormality is alleviated bydecreasing the activity of a mammalian NPFF receptor which comprisesadministering to the subject an effective amount of the pharmaceuticalcomposition of claim 122, thereby treating the abnormality.
 124. Amethod for identifying an agonist capable of alleviating an abnormalityin a subject wherein the abnormality is alleviated by increasing theactivity of a mammalian NPFF receptor comprising administering acompound to the transgenic, nonhuman mammal of claim 70, 73, 74, or 75,and determining whether the compound alleviates the physical andbehavioral abnormalities displayed by the transgenic, nonhuman mammal,the alleviation of the abnormality identifying the compound as anagonist.
 125. The method of claim 124, wherein the mammalian NPFFreceptor is a human NPFF1 receptor.
 126. The method of claim 124,wherein the mammalian NPFF receptor is a human NPFF2 receptor.
 127. Anagonist identified by the method of claim
 124. 128. A pharmaceuticalcomposition comprising an agonist identified by the method of claim 127and a pharmaceutically acceptable carrier.
 129. A method of treating anabnormality in a subject wherein the abnormality is alleviated byincreasing the activity of a mammalian NPFF receptor which comprisesadministering to the subject an effective amount of the pharmaceuticalcomposition of claim 128, thereby treating the abnormality.
 130. Amethod for diagnosing a predisposition to a disorder associated with theactivity of a specific mammalian allele which comprises: (a) obtainingDNA of subjects suffering from the disorder; (b) performing arestriction digest of the DNA with a panel of restriction enzymes; (c)electrophoretically separating the resulting DNA fragments on a sizinggel; (d) contacting the resulting gel with a nucleic acid probe capableof specifically hybridizing with a unique sequence included within thesequence of a nucleic acid molecule encoding a mammalian NPFF receptorand labeled with a detectable marker; (e) detecting labeled bands whichhave hybridized to the DNA encoding a mammalian NPFF1 receptor of claim1 labeled with a detectable marker to create a unique band patternspecific to the DNA of subjects suffering from the disorder; (f)preparing DNA obtained for diagnosis by steps (a)-(e); and (g) comparingthe unique band pattern specific to the DNA of subjects suffering fromthe disorder from step (e) and the DNA obtained for diagnosis from step(f) to determine whether the patterns are the same or different and todiagnose thereby predisposition to the disorder if the patterns are thesame.
 131. The method of claim 130, wherein a disorder associated withthe activity of a specific mammalian allele is diagnosed.
 132. A methodof preparing the purified mammalian NPFF receptor of claim 23 whichcomprises: (a) culturing cells which express the mammalian NPFFreceptor; (b) recovering the mammalian NPFF receptor from the cells; and(c) purifying the mammalian NPFF receptor so recovered.
 133. A method ofpreparing the purified mammalian NPFF receptor of claim 23 whichcomprises: (a) inserting a nucleic acid encoding the mammalian NPFFreceptor into a suitable vector; (b) introducing the resulting vectorinto a suitable host cell; (c) placing the resulting cell in suitablecondition permitting the production of the mammalian NPFF receptor; (d)recovering the mammalian NPFF receptor produced by the resulting cell;and (e) isolating and/or purifying the mammalian NPFF receptor sorecovered.
 134. A process for determining whether a chemical compound isa mammalian NPFF receptor agonist which comprises contacting cellstransfected with and expressing DNA encoding the mammalian NPFF receptorwith the compound under conditions permitting the activation of themammalian NPFF receptor, and detecting an increase in mammalian NPFFreceptor activity, so as to thereby determine whether the compound is amammalian NPFF receptor agonist.
 135. A process for determining whethera chemical compound is a mammalian NPFF receptor antagonist whichcomprises contacting cells transfected with and expressing DNA encodingthe mammalian NPFF receptor with the compound in the presence of a knownmammalian NPFF receptor agonist, under conditions permitting theactivation of the mammalian NPFF receptor, and detecting a decrease inmammalian NPFF receptor activity, so as to thereby determine whether thecompound is a mammalian NPFF receptor antagonist.
 136. A process ofclaim 134 or 135, wherein the mammalian NPFF receptor is a human NPFF1receptor.
 137. A process of claim 134 or 135, wherein the mammalian NPFFreceptor is a human NPFF2 receptor.
 138. A pharmaceutical compositionwhich comprises an amount of a mammalian NPFF receptor agonistdetermined by the process of claim 134 effective to increase activity ofa mammalian NPFF receptor and a pharmaceutically acceptable carrier.139. A pharmaceutical composition of claim 138, wherein the mammalianNPFF receptor agonist is not previously known.
 140. A pharmaceuticalcomposition which comprises an amount of a mammalian NPFF receptorantagonist determined by the process of claim 135 effective to reduceactivity of a mammalian NPFF receptor and a pharmaceutically acceptablecarrier.
 141. A pharmaceutical composition of claim 140, wherein themammalian NPFF receptor antagonist is not previously known.
 142. Aprocess for determining whether a chemical compound specifically bindsto and activates a mammalian NPFF receptor, which comprises contactingcells producing a second messenger response and expressing on their cellsurface the mammalian NPFF receptor, wherein such cells do not normallyexpress the mammalian NPFF receptor, with the chemical compound underconditions suitable for activation of the mammalian NPFF receptor, andmeasuring the second messenger response in the presence and in theabsence of the chemical compound, a change in the second messengerresponse in the presence of the chemical compound indicating that thecompound activates the mammalian NPFF receptor.
 143. The process ofclaim 142, wherein the second messenger response comprises chloridechannel activation and the change in second messenger is an increase inthe level of inward chloride current.
 144. A process for determiningwhether a chemical compound specifically binds to and inhibitsactivation of a mammalian NPFF receptor, which comprises separatelycontacting cells producing a second messenger response and expressing ontheir cell surface the mammalian NPFF receptor, wherein such cells donot normally express the mammalian NPFF receptor, with both the chemicalcompound and a second chemical compound known to activate the mammalianNPFF receptor, and with only the second chemical compound, underconditions suitable for activation of the mammalian NPFF receptor, andmeasuring the second messenger response in the presence of only thesecond chemical compound and in the presence of both the second chemicalcompound and the chemical compound, a smaller change in the secondmessenger response in the presence of both the chemical compound and thesecond chemical compound than in the presence of only the secondchemical compound indicating that the chemical compound inhibitsactivation of the mammalian NPFF receptor.
 145. The process of claim144, wherein the second messenger response comprises chloride channelactivation and the change in second messenger response is a smallerincrease in the level of inward chloride current in the presence of boththe chemical compound and the second chemical compound than in thepresence of only the second chemical compound.
 146. A process of any ofclaims 142, 143, 144, or 145, wherein the mammalian NPFF receptor is ahuman NPFF1 receptor.
 147. A process of any of claims 142, 143, 144, or145, wherein the mammalian NPFF receptor is a human NPFF2 receptor. 148.The process of any of claims 142, 143, 144, 145, 146, or 147 wherein thecell is an insect cell.
 149. The process of any of claims 142, 143, 144,145, 146, or 147, wherein the cell is a mammalian cell.
 150. The processof claim 149, wherein the mammalian cell is nonneuronal in origin. 151.The process of claim 150, wherein the nonneuronal cell is a COS-7 cell,CHO cell, 293 human embryonic kidney cell, NIH-3T3 cell or LM(tk-) cell.152. The process of claim 142, 143, 144, or 145 wherein the compound isnot previously known to bind to a mammalian NPFF receptor.
 153. Acompound determined by the process of claim
 152. 154. A pharmaceuticalcomposition which comprises an amount of a mammalian NPFF receptoragonist determined by the process of claim 142 or 143 effective toincrease activity of a mammalian NPFF receptor and a pharmaceuticallyacceptable carrier.
 155. A pharmaceutical composition of claim 154,wherein the mammalian NPFF receptor agonist is not previously known.156. A pharmaceutical composition which comprises an amount of amammalian NPFF receptor antagonist determined by the process of claim144 or 145 effective to reduce activity of a mammalian NPFF receptor anda pharmaceutically acceptable carrier.
 157. A pharmaceutical compositionof claim 156, wherein the mammalian NPFF receptor antagonist is notpreviously known.
 158. A method of screening a plurality of chemicalcompounds not known to activate a mammalian NPFF receptor to identify acompound which activates the mammalian NPFF receptor which comprises:(a) contacting cells transfected with and expressing the mammalian NPFFreceptor with the plurality of compounds not known to activate themammalian NPFF receptor, under conditions permitting activation of themammalian NPFF receptor; (b) determining whether the activity of themammalian NPFF receptor is increased in the presence of the compounds;and if so (c) separately determining whether the activation of themammalian NPFF receptor is increased by each compound included in theplurality of compounds, so as to thereby identify the compound whichactivates the mammalian NPFF receptor.
 159. A method of claim 158,wherein the mammalian NPFF receptor is a human NPFF1 receptor.
 160. Amethod of claim 158, wherein the mammlian NPFF receptor is a human NPFF2receptor.
 161. A method of screening a plurality of chemical compoundsnot known to inhibit the activation of a mammalian NPFF receptor toidentify a compound which inhibits the activation of the mammalian NPFFreceptor, which comprises: (a) contacting cells transfected with andexpressing the mammalian NPFF receptor with the plurality of compoundsin the presence of a known mammalian NPFF receptor agonist, underconditions permitting activation of the mammalian NPFF receptor; (b)determining whether the activation of the mammalian NPFF receptor isreduced in the presence of the plurality of compounds, relative to theactivation of the mammalian NPFF receptor in the absence of theplurality of compounds; and if so (c) separately determining theinhibition of activation of the mammalian NPFF receptor for eachcompound included in the plurality of compounds, so as to therebyidentify the compound which inhibits the activation of the mammalianNPFF receptor.
 162. A method of claim 161, wherein the mammalian NPFFreceptor is a human NPFF1 receptor.
 163. A method of claim 161, whereinthe mammalian NPFF receptor is a human NPFF2 receptor.
 164. A method ofany of claims 158, 159, 160, 161, 162, or 163, wherein the cell is amammalian cell.
 165. A method of claim 164, wherein the mammalian cellis non-neuronal in origin.
 166. The method of claim 165, wherein thenon-neuronal cell is a COS-7 cell, a 293 human embryonic kidney cell, aLM(tk-) cell or an NIH-3T3 cell.
 167. A pharmaceutical compositioncomprising a compound identified by the method of claim 158 or 159effective to increase mammalian NPFF receptor activity and apharmaceutically acceptable carrier.
 168. A pharmaceutical compositioncomprising a compound identified by the method of claim 161 or 162effective to decrease mammalian NPFF receptor activity and apharmaceutically acceptable carrier.
 169. A method of treating anabnormality in a subject wherein the abnormality is alleviated byincreasing the activity of a mammalian NPFF receptor which comprisesadministering to the subject an amount of a compound which is amammalian NPFF receptor agonist effective to treat the abnormality. 170.A method of claim 169, wherein the abnormality is a regulation of asteroid hormone disorder, an epinephrine release disorder, agastrointestinal disorder, a cardiovascular disorder, an electrolytebalance disorder, hypertension, diabetes, a respiratory disorder,asthma, a reproductive function disorder, an immune disorder, anendocrine disorder a musculoskeletal disorder, a neuroendocrinedisorder, a cognitive disorder, a memory disorder, a sensory modulationand transmission disorder, a motor coordination disorder, a sensoryintegration disorder, a motor integration disorder, a dopaminergicfunction disorder, an appetite disorder, obesity, a sensory transmissiondisorder, an olfaction disorder, a sympathetic innervation disorder,pain, psychotic behavior, morphine tolerance, opiate addiction,affective disorder, or migraine.
 171. A method of treating anabnormality in a subject wherein the abnormality is alleviated bydecreasing the activity of a mammalian NPFF receptor which comprisesadministering to the subject an amount of a compound which is amammalian NPFF receptor antagonist effective to treat the abnormality.172. A method of claim 171, wherein the abnormality is a regulation ofsteroid hormone disorder, an epinephrine release disorder, agastrointestinal disorder, a cardiovascular disorder, an electrolytebalance disorder, hypertension, diabetes, a respiratory disorder,asthma, a reproductive function disorder, an immune disorder, anendocrine disorder, a musculoskeletal disorder, a neuroendocrinedisorder, a cognitive disorder, a memory disorder, a sensory modulationand transmission disorder, a motor coordination disorders a sensoryintegration disorder, a motor integration disorder, a dopaminergicfunction disorder, an appetite disorder, obesity, a sensory transmissiondisorder, an olfaction disorder, a sympathetic innervation disorder,pain, psychotic behavior, morphine tolerance, opiate addiction,affective disorder, or migraine.
 173. A process for making a compositionof matter which specifically binds to a mammalian NPFF receptor whichcomprises identifying a chemical compound using the process of any ofclaims 76, 77, 97, 98, 107, or 108 and then synthesizing the chemicalcompound or a novel structural and functional analog or homolog thereof.174. A process for making a composition of matter which specificallybinds to a mammalian NPFF receptor which comprises identifying achemical compound using the process of any of claims 134, 142, or 158and then synthesizing the chemical compound or a novel structural andfunctional analog or homolog thereof.
 175. A process for making acomposition of matter which specifically binds to a mammalian NPFFreceptor which comprises identifying a chemical compound using theprocess of any of claims 135, 144, 161 and then synthesizing thechemical compound or a novel structural and functional analog or homologthereof.
 176. The process of any of claims 173, 174, or 175, wherein themammalian NPFF receptor is a human NPFF1 receptor.
 177. The process ofany of claims 173, 174, or 175, wherein the mammalian NPFF receptor is ahuman NPFF2 receptor.
 178. A process for preparing a pharmaceuticalcomposition which comprises admixing a pharmaceutically acceptablecarrier and a pharmaceutically acceptable amount of a chemical compoundidentified by the process of any of claims 76, 77, 97, 98, 107, or 108or a novel structural and functional analog or homolog thereof .
 179. Aprocess for preparing a pharmaceutical composition which comprisesadmixing a pharmaceutically acceptable carrier and a pharmaceuticallyacceptable amount of a chemical compound identified by the process ofany of claims 134, 142, or 158 or a novel structural and functionalanalog or homolog thereof.
 180. A process for preparing a pharmaceuticalcomposition which comprises admixing a pharmaceutically acceptablecarrier and a pharmaceutically acceptable amount of a chemical compoundidentified by the process of any of claims 135, 144, or 161 or a novelstructural and functional analog or homolog thereof.
 181. The process ofany of claims 178, 179, or 180, wherein the mammalian NPFF receptor is ahuman NPFF1 receptor.
 182. The process of any of claims 178, 179, or180, wherein the mammalian NPFF receptor is a human NPFF2 receptor.